miRNA FOR TREATING CANCER AND FOR USE WITH ADOPTIVE IMMUNOTHERAPIES

ABSTRACT

In some aspects, miRNA for the treatment of cancer are provided. In some embodiments, a miRNA (e.g., miR-124, miR-142, and/or miR-138) may be used to promote or enhance immune destruction of a cancer, or reduce the immune suppression of the cancer, in a subject. In other aspects, the miRNA may be used in, or in combination with, an adoptive immunotherapy.

This application is a divisional of U.S. application Ser. No.14/775,667, filed Sep. 11, 2015, which is a national phase applicationunder 35 U.S.C. §371 of International Application No. PCT/US2014/028296,filed Mar. 14, 2014, which claims the benefit of U.S. Provisional PatentApplication No. 61/782,921, filed Mar. 14, 2013, the entirety of each ofwhich is incorporated herein by reference.

The invention was made with government support under grantsRO1-CA1208113, P50-CA127001, and P50-CA093459 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the field of molecularbiology and medicine. More particularly, it concerns miRNA for thetreatment of cancer and their use as an immunotherapeutic.

2. Description of Related Art

Glioblastoma, the most common type of primary malignant brain tumor, isassociated with disproportionately high morbidity and mortality. Despitemulti-modality treatment, the median survival time is 14.6 months forpatients with newly-diagnosed glioblastoma (Stupp et al., 2005).Significant and profound immunosuppression exists in the glioblastomamicroenvironment and systemically that participates in the glioblastomapathophysiology by both inhibiting anti-tumor immunity and promotingglioma invasion and progress. Specifically, it has been shown thattumor-associated macrophages, the largest infiltrating immune cellpopulation in glioblastomas (Hussain et al., 2006), do not participatein anti-tumor immune responses but rather support the glioblastomainvasion, progression, and therapeutic resistance (Wu et al., 2010).Micro RNAs (miRNA or miRs) are non-coding molecules involved inpost-transcriptional gene regulation that have been shown to modulatetumor cell proliferation and apoptosis and to act as oncogenes ortumor-suppressor genes (Gabriely et al., 2008; Iliopoulos et al., 2010).Although some miRNA have been linked to tumor progression, theconnection between tumor-mediated immune modulation and miRNA has notbeen previously demonstrated. Clearly, there is a need for newtreatments for cancers, such as glioblastoma.

SUMMARY OF THE INVENTION

The present invention overcomes limitations in the prior art byexploiting the immune system to mediate the anti-tumor effects of miRNAfor the treatment of cancer or inflammation. In some aspects, miR-124(SEQ ID NO:1), miR-142-3p (SEQ ID NO:2), and/or miR-138 (SEQ ID NO:3)may be administered to a subject, such as a human patient, to treat acancer, such as a glioblastoma. In some embodiments, the miRNA maypromote activation of the immune system and as such may be used as animmunotherapeutic. As shown in the below examples, in vivo local orsystemic administration of miR-124, miR-142-3p or miR-138 resulted ininhibition of glioma growth and extended survival in a glioma mousemodel. For example, as shown in the below examples, in vivo treatment ofestablished subcutaneous glioma cells with miR-138 demonstrated thatgliomas started to shrink as soon as miR-138 was intravenouslyadministered to mice; further, the gliomas continued to regress evenafter miR-138 treatment was discontinued.

An aspect of the present invention relates to a method of inducing ananti-cancer immune response in a subject, comprising administering toimmune cells of said subject, or contacting the immune cells of thesubject with, a pharmaceutically effective amount of a nucleic acidcomposition comprising a miR-124, a miR-142, or a miR-138 nucleic acidsequence in an amount sufficient to induce, enhance, or promote animmune response against the cancer in the subject. The immune cells maycomprise T-cells, natural killer (NK) cells, or dendritic cells. Theimmune cells may be contacted in vivo. In some embodiments, the nucleicacid composition is administered parenterally to the subject. Forexample, the nucleic acid composition may be administered to the subjectintradermally, intravenously, intraarterially, intrathecally,intraperitoneally, intramuscularly, or by injection into asurgical/resection cavity. The nucleic acid composition may beadministered to the subject via an aerosol. In some embodiments, theimmune cells are contacted with the nucleic acid composition ex vivo inan amount sufficient to immunologically prime the immune cells, and theimmunologically primed immune cells are subsequently administered to thepatient. In some embodiments, miR-124 is administered to the immunecells. In some embodiments, miR-142 is administered to the immune cells.In some embodiments, miR-138 is administered to the immune cells. Thenucleic acid may be a modified nucleic acid such as, e.g., a LNA. Insome embodiments, the nucleic acid is an unmodified nucleic acid. Thecancer may be selected from the group consisting of a brain cancer, aglioma, a neuroblastoma, a medulloblastoma, a glioblastoma, anastrocytoma, or a melanoma. In some embodiments, the cancer is a braincancer, a glioma, a neuroblastoma, or glioblastoma. The nucleic acid maycomprise a phosphoramidate linkage, a phosphorothioate linkage, aphosphorodithioate linkage, or an O-methylphosphoroamidite linkage. Thenucleic acid may comprise one or more nucleotide analogs. The subject isa mammal such as, e.g., a human. In some embodiments, the method furthercomprises administering to the subject a chemotherapy, immunotherapy,radiotherapy, cytokine therapy, or surgery. In some embodiments, animmunotherapy is administered to the subject. The immunotherapy may bean adoptive immunotherapy. The adoptive immunotherapy may comprise aT-cell immunotherapy, a natural killer (NK) cell immunotherapy, adendritic cell immunotherapy, a viral immunotherapy, or an adoptiveT-cell transfer. The immunotherapy may comprise administration of amonoclonal antibody, interleukin 2 (IL-2), or gamma interferon to thesubject. In some embodiments, a monoclonal antibody is administered tothe subject, wherein the monoclonal antibody selectively targets animmune checkpoint or immune suppressive pathway or mechanism. Thenucleic acid may be comprised in a vector such as, e.g., a viral vector.The viral vector may be an adenovirus, an adeno-associated virus, alentivirus, or a herpes virus. In some embodiments, the vector comprisesa lipid, lipid emulsion, liposome, nanoparticle, or exosomes. The miRNAmay be comprised in a liposome, nanoparticle, or exosome. The miRNA maybe comprised in a liposome, wherein the liposome comprisesN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP) or lipofectamine′. Alternately, the liposome or nanoparticle maycomprise chitosan, cholesterol, (DOTAP and cholesterol), polyethyleneglycol (PEG), dimyristoyl-phosphatidylcholine (DMPC),dimyristoylphosphatidylglycerol (DMPG), soy phosphatidylcholine (HSPC),cholesterol, phosphatidylglycerol (DSPG), dioleoylphosphatidylcholine(DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), egg lecithin,MPEG-DSPE, Soybean oil, Polysorbate 80, or egg sphingomyelin. The miRNAmay be comprised in a nanoparticle, wherein the nanoparticle comprisessilicone or gold.

Another aspect of the present invention relates to a pharmaceuticalpreparation comprising miR-124, miR-142, or miR-138. The miRNA maycomprise a phosphoramidate linkage, a phosphorothioate linkage, aphosphorodithioate linkage, or an O-methylphosphoroamidite linkage. Insome embodiments, the miRNA is a LNA. The pharmaceutical preparation maybe formulated for intravenous, intraperitoneal, intratumoral,intrathecal, intranasal, intralymphatic, or oral administration. ThemiRNA may be comprised in a liposome, nanoparticle, or exosome. Thepharmaceutical preparation may compriseN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP) or Lipofectamine™.

Yet another aspect of the present invention relates to a method oftreating a cancer in an individual, comprising: (a) contacting T-cells,natural killer (NK) cells, or dendritic cells to be used in an adoptivetherapy with a synthetic or recombinant miR-124, miR-142, or miR-138 inan amount sufficient to promote or enhance the function or proliferationof the cells; and (b) administering the T-cells, natural killer (NK)cells, or dendritic cells to the individual. The cells may be incubatedin the presence of the miRNA in vitro, wherein the miRNA are syntheticor recombinant miRNA. The miRNA may be comprised in a liposome during atleast a portion of said incubation. The liposome may compriseN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP) or Lipofectamine™. The miRNA may be encoded by a nucleic acid,wherein the nucleic acid has been transfected into the cells. Saidtransfection may comprise electroporation or incubation with a viralvector. The viral vector may be an adenovirus, an adeno-associatedvirus, a lentivirus, or a herpes virus.

In some embodiments, miRNAs may be combined (e.g., miR-124, miR-142-3p,and/or miR-138) for further therapeutic synergy and/or comprehensivetargeting of tumor-mediated immune suppression. In some embodiments, thecombined miRNAs may be administered to a subject, such as a humanpatient, in combination with one or more additional immune therapy. Forexample, in some aspects a pharmaceutical preparation may comprise(miR-124 and miR-142-3p), (miR-124 and miR-138), (miR-142-3p andmiR-138), or (miR-124, miR-142-3p, and miR-138) and an excipient. Thepharmaceutical preparation may be formulated in a liposome or otherpharmaceutical preparation as described herein. The pharmaceuticalpreparation may be used to treat a cancer (e.g., a glioma, etc.) asdescribed herein. The combination therapy of a miRNA may be furthercombined with an additional therapy such as, e.g., a radiation therapy,surgery, additional chemotherapy (e.g., a STAT3 inhibitor such asWP1066, etc.), or additional immune therapy to treat a cancer (e.g., aglioma, etc.) as described herein.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-E. miR-124 expression is significantly reduced in glioblastomaand inhibits the signal transducer and activator of transcription 3(STAT3). (FIG. 1A) Relative expression levels of miR-124 were detectedby TaqMan quantitative PCR. There was a significant difference in therelative expression levels of miR-124 between normal brain tissues,glioblastoma specimens, and glioma cancer stem cells (gCSCs). (FIG. 1B)Representative specimens of normal brain (I) demonstrating miR-124expression (arrow) by in situ hybridization in neurons and in aglioblastoma (II) lacking miR-124 expression (at 40× magnification).(FIG. 1C) Sequence of the predicted miR-124 binding site (SEQ ID NO:24)on the STAT3-3′UTR (SEQ ID NO:25) and the STAT3-3′UTR mutant (SEQ IDNO:26) that disrupted miR-124 binding. On the basis of predictivealgorithms, there exists an 11-bp nucleotide binding site in the STAT33′-UTR that is imperfectly paired with miR-124, which is predicted tolead to STAT3 mRNA degradation. (FIG. 1D) Relative luciferase activityof HeLa cells after transfection with miR-124- or scramblecontrol-expressing plasmids in conjunction with a wild type STAT3 3′-UTRor miR-124 binding site mutant reporter construct, demonstratinginhibition of STAT3. **P<0.01. (FIG. 1E) Western blot analysis of theSTAT3 signaling pathway gCSCs transfected with scramble control (S)compared with miR-124 (M).

FIGS. 2A-E. miR-124 reverses glioma-mediated immunosuppression. (FIG.2A) The gCSC phenotype was markedly altered by miR-124, as photographed48 hours after transfection. (FIG. 2B) T-cell proliferation was detectedby flow cytometry analysis with CFSE staining after 3 days of treatmentwith medium alone, gCSC-scramble-transfected conditioned medium,gCSC-miR-124-transfected conditioned medium, orgCSC-miR-124+STAT3-transfected conditioned medium. miR-124 up regulationcaused a reversal of inhibition in T-cell proliferation compared withthe gCSC-scramble-transfected conditioned medium. STAT3 overexpressionrestored gCSC inhibition on T-cell proliferation. (FIG. 2C) T-cellapoptosis was measured by the percentage of annexin V+7-AAD+ cells.Similarly, miR-124 up regulation decreased gCSC-induced T-cellapoptosis, whereas STAT3 overexpression reversed the miR-124 effect.(FIG. 2D) T-cells were analyzed on the basis of CD4 and FoxP3 expressionlevels, by flow cytometry analysis. Up regulation of miR-124 caused adecline in Treg induction compared with the scrambled gCSC control, andSTAT3 addition restored the ability of the gCSCs to induce Tregs. (FIG.2E) The functional suppressive activity of FoxP3+ Tregs induced in (FIG.2D) was verified by autologous coculture with CD4+ T-cells. Theexperiments were repeated three times with similar results, and onerepresentative set of data is shown.

FIGS. 3A-D. miR-124 enhances T-cell effector cytokine production inglioblastoma patients. PBMCs were isolated from the freshly drawn bloodof glioblastoma patients undergoing resection, transfected with miR-124or scramble control oligonucleotides, and sequentially stimulated withanti-CD3/anti-CD28 antibodies for T-cell proliferation. miR-124administration significantly enhanced effector cytokine responses, suchas IFN-γ (n=10) (FIG. 3A), IL-2 (n=10) (FIG. 3B), and TNF-α (n=7) (FIG.3C), in both CD4+ T and CD8+ T-cells of PBMCs from glioblastomapatients. miR-124 up regulation suppressed pSTAT3 activity in CD8+T-cells (n=9) and to a lesser degree in the CD4+ T-cells (n=9) (FIG.3D). Each symbol represents PBMCs from different patients withouttransfection or with transfection with either the scramble control ormiR-124.

FIGS. 4A-F. miR-124 exerts potent efficacy to suppress subcutaneousGL261 tumors in a syngeneic mouse model. (FIG. 4A) The treatment schemaand the volumes of subcutaneous GL261 tumors in C57BL/6J mice treatedintratumorally with either miR-124 or scramble control, or leftuntreated starting on day 6 (n=10/group/experiment). In the miR-124group, * denotes a P value of <0.01 compared with both the untreated andscramble control tumors. Standard deviations are shown. Arrows indicatedays of treatment and tumor size measurements. The inset photo on theright is of a representative ex vivo GL261 tumor comparison betweenmiR-124- and scramble control-treated tumor-bearing mice. (FIG. 4B) Exvivo immunohistochemical analysis of gliomas, untreated (n=2), ortreated with a scramble control (n=5), or miR-124 (n=3), whichdemonstrates a marked decrease in p-STAT3 expression in the local tumormicroenvironment (P=0.0039). (FIG. 4C) Splenocytes from miR-124intratumorally treated GL261 mice (n=3) have markedly increasedcytotoxicity at 48 hours after coculture with GL261 cells compared withthat in splenocytes from scramble control oligonucleotide-treatedtumor-bearing mice (P<0.001). The ratios of splenocytes to GL261 cellsare 10:1, 40:1, and 100:1. Error bars in the curve represent thestandard deviation in the data from 3 mice. (FIG. 4D) Decreasedtumor-infiltrating FoxP3+ Tregs in miR-124-treated GL261 mice. A singleexample is shown, but identical results were obtained in two other tumorbearing animals. Furthermore, miR-124 administration enhances IFN-γ andTNF-α production in CD4 T-cells (FIG. 4E) and CD8 T-cells (FIG. 4F) inthe tumor local microenvironment. One set of representative FACS plotsis shown. Similar results were obtained from another two tumor-bearingmice treated with miR-124 or scramble control, respectively.

FIGS. 5A-D. The therapeutic effect of miR-124 is lost inimmune-incompetent models. (FIG. 5A) The treatment schema and thevolumes of subcutaneous GL261 tumors in C57BL/6J mice treatedintravenously with miR-124 or scramble control starting on day 7(n=10/group/experiment). In the miR-124 group, * denotes a P value of<0.01 compared with scramble control tumors. Standard deviations areshown. The arrows indicate days of treatment and tumor sizemeasurements. (FIG. 5B) The treatment schema and volumes of subcutaneousGL261 tumors in nude mice treated intratumorally with miR-124 orscramble control starting on day 7 (n=10/group/experiment). Standarddeviations are shown. Arrows indicate days of treatment and tumor sizemeasurements. (FIG. 5C) Treatment schema and graph of the Kaplan-Meierestimate, demonstrating better survival in C57BL/6J mice withmiR-124-treated intracerebral GL261 gliomas (n=10/group/experiment) thanin scrambled controls. * denotes a P value=0.02 compared with scramblecontrol gliomas. (FIG. 5D) Graph of the Kaplan-Meier estimatedemonstrates miR-124's lack of therapeutic effect in nude mice withintracerebral GL261 gliomas (n=10/group/experiment, P=0.24).

FIGS. 6A-C. T-cells mediate the antiglioma immune therapeutic efficacyof miR-124. (FIG. 6A) Histogram shows miR-124 transfection inhibitedp-STAT3 activity in adoptively transferred T cells. (FIG. 6B) Thetreatment schema and volumes of subcutaneous GL261 tumors in C57BL/6Jmice treated intratumorally with miR-124 or scramble controlsubcutaneously on day 7 (n=8/group/experiment) in the setting of in vivoCD4+ T-cells or CD8+ T-cell depletion. Arrows indicate days of treatmentand tumor size measurements. *denotes a P value of <0.01 comparing theanti-CD4 or anti-CD8 depletion group with the isotype andmiR-124-treated group. Standard deviations are shown. (FIG. 6C) Thetreatment schema and volumes of subcutaneous GL261 tumors in C57BL/6Jmice treated intravenously with miR-124 or scramble control transfectedCD3+ T-cells on day 14 (n=10/group/experiment). *denotes a P value of<0.01 comparing the miR-124 transfected T-cell treated group with thescramble control and untreated group.

FIGS. 7A-E. miR-124 exerts a therapeutic effect in Ntv-a mice. (FIG. 7A)Representative hematoxylin and eosin staining of a high-grade gliomainduced in Ntv-a mice transfected with RCAS-PDGFB and RCAS-STAT3transgenes demonstrates neovascular proliferation (arrow) andpseudopallisading necrosis (arrowhead) at 100× magnification. (FIG. 7B)Representative specimen from the brain of an Ntv-a mouse transfectedwith the RCAS-PDGFB and RCAS-STAT3 transgenes demonstrates miR-124expression by in situ hybridization in neurons surrounding a gliomadevoid of miR-124 expression (arrow) at 400× magnification. (FIG. 7C)Treatment schema and graph of the Kaplan-Meier estimate demonstratesimproved survival in miR-124-treated Ntv-a mice transfected with theRCAS-PDGFB and RCAS-STAT3 transgenes (n=9/group) compared with scramblecontrol and untreated mice (lipofectamine 2000 vehicle only). *denotes aP value of 0.04. (FIG. 7D) Summary graph demonstrates the incidence ofhigh- and low-grade gliomas on the basis of hematoxylin and eosinstaining features of necrosis and neovascular proliferation inmiR-124-treated Ntv-a mice transfected with RCAS-PDGFB and RCAS-STAT3transgenes (n=7) compared with scrambled controls (n=8) and untreatedmice (n=7) (P<0.0001). (FIG. 7E) An ex vivo immunohistochemical analysisof gliomas, untreated (n=7) or treated with a scramble control (n=6) ormiR-124 (n=7), demonstrates a marked decrease in p-STAT3 expression inthe local tumor microenvironment of the miR-124-treated group (scramblevs. miR-124: P=0.003; untreated vs miR-124: P=0.007; untreated vsscramble: P=0.87). Quantification of p-STAT3 expression was obtained byaveraging the number of nuclear positive p-STAT3 cells byimmunohistochemistry from 10 non-overlapping high-power microscopicfields (magnification×400) of the gliomas obtained from either untreatedRCAS-PDGF-B+RCAS-STAT3 mice or mice treated with the scramble control ormiR-124. Each dot represents the analysis of one mouse glioma.

FIGS. 8A-B. (FIG. 8A) Summary dot plot demonstrating miR-124 expressionis induced in gCSCs (n=8) upon neural differentiation (P<0.01). Blackfilled circles: gCSCs; black filled squares: differentiated gCSCs. (FIG.8B) Interleukin-8 (IL-8) (P<0.05), galectin-3 (P<0.01), and MIC-1(P<0.05) produced by gCSCs were reduced upon transfection with miR-124compared with their levels in the scramble control.

FIG. 9. Schema demonstrating how miR-124 can exert target effects on theSTAT3 pathway in gCSC cells. STAT3 can be activated by a variety ofligands such as IL-6 or EGFR. miR-124 can not only down-modulate SHC1,STAT3, P-STAT3 in all tested gCSC cell lines but also, occasionally,pMAPK1/3, probably in a contextual fashion when the IL-6R ligand is notpresent and the gCSC is dependent on EGFR signaling.

FIGS. 10A-C. miR-21 expression is higher in gCSCs and glioblastomatumors than in normal brain and is inhibited by miR-124 overexpression.(FIG. 10A) Relative expression levels of miR-21 were detected by TaqManquantitative PCR. There were significant differences in the relativeexpression levels of miR-21 between normal brain tissues, gCSCs andglioblastoma specimens. (FIG. 10B) T-cell proliferation was detected byflow cytometry analysis with CFSE staining after 3 days of treatmentwith medium alone, gCSC-scramble-transfected conditioned medium, orgCSC-miR-21-transfected conditioned medium. miR-21 up regulation causedfurther inhibition in T-cell proliferation compared with thegCSC-scramble-transfected conditioned medium. (FIG. 10C) Forcedoverexpression of miR-124 in the gCSCs resulted in the down modulationof miR-21 expression.

FIG. 11. Ex vivo splenocytes from miR-124 treated intracranial GL261mice have markedly increased cytotoxicity at 48 hours after coculturewith GL261 cells compared to splenocytes from scramble controloligonucleotide-treated GL261-bearing mice. The ratio of splenocytes toGL261 cells is 100:1. Representative dot plots of duel PI- andCFSE-labeled GL261 cells with splenocytes and the associated gatingstrategy are shown to demonstrate how the target cells (GL261) areidentified and quantified.

FIGS. 12A-C. Up regulation of miR-124 level and down regulation ofp-STAT3 in peripheral blood T-cells and glioma-infiltrating T-cellsafter intravenous administration of miR-124. (FIG. 12A). The miR-124expression level is below the limit of detection in CD3+ T-cellsisolated from the blood of non-tumor bearing C57/BL6J mice (n=3) andGL261-bearing mice (n=3) (P>0.05). Levels are shown relative to U6snRNA. (FIG. 12B) miR-124 is detected in both CD3+ T-cells isolated fromblood (P<0.05 relative to scramble control; n=3) and gliomas (P<0.05relative to scramble control; n=3) 18 hours after the in vivoadministration of miR-124. (FIG. 12C) Coinciding with the presence ofmiR-124, p-STAT3 expression levels were decreased in the T-cells fromthe peripheral blood and gliomas in the miR-124 treated mice.Representative histograms were shown. The p-STAT3 expression levelsrelative to isotype control in the peripheral blood T-cells in themiR-124 treated mice (1.4±0.2%) was down-modulated compared to scrambletreated mice (17.9±1.9%) (P=0.007; n=3) and glioma-infiltrating T-cells(miR-124: 4.3±0.1%; scramble: 18.2±4.3%; P=0.007), but not within thesplenic T-cells (miR-124: 16.8±0.9%; scramble: 12.9±2.7%; P=0.07).

FIG. 13. miR-124 modulates Th1, Th17, and inducible Tregdifferentiation. CD4+CD45RA+CD45RO-naïve T-cells were isolated fromhealthy donor PBMCs and stimulated with plate-bound anti-CD3 and solubleanti-CD28 under Th1, Th17, and inducible Treg polarization conditionsbefore miR-124 transfection. After 3 rounds of T-cell stimulation andpolarization, cytokine production in the different Th populations wasquantified by intracellular cytokine FACS. One representative set ofdata was shown, but similar results were obtained from two additionalindependent experiments.

FIGS. 14A-B. miR-142-3p Expression in Gliomas. (FIG. 14A). Heatmapsdemonstrating the miRNA expression pattern in glioblastoma-infiltratingmacrophages compared with matched peripheral blood monocytes, utilizingthe Human miRNA OneArray Microarray v2. With a mean 4.9-fold decrease inlevel relative to matched peripheral blood monocytes, miR-142-3p emergedas a leading down-regulated candidate. (FIG. 14B) Total RNA wasextracted from gCSCs (n=5), glioma cell lines (U-87 and U-251),glioblastomas (n=4), healthy donor peripheral blood CD14+ monocytes(n=3), glioblastoma patient peripheral blood CD14+ monocytes (n=6), andglioblastoma infiltrating CD11b+ macrophages (n=3). Analysis byquantitative reverse transcription polymerase chain reactiondemonstrated that although miR-142-3p is not expressed in glioma celllines or gCSCs, monocytes express high levels of miR-142-3p and are amajor contributing source of miR-142-3p in glioblastomas.

FIGS. 15A-D. Preferential Expression of miR-142-3p in M1 Macrophages.Human CD14+ monocytes were incubated with GM-CSF or M-CSF to induce M1and M2 macrophages, respectively. FIG. 15A, The M1 and M2 macrophageshave a distinctive in vitro morphology. Scale bar=20 μM. FIG. 15B, Cellsurface expression of CD14, CD45, CD11b, CD80, CD86, MHC II, CD68 andCD163 were evaluated by flow cytometry. Representative results areshown. Similar result was observed in 3 replicates. The grey histogramdenotes M1 macrophages, black denotes M2 macrophages, and the dottedline represents the isotype control. FIG. 15C, The phagocytic activityof M1 and M2 was measured by fluorescent uptake and summarized. Errorbars represent standard deviations. A paired two-sided Student t testwas used. n=6, ***P<0.001. FIG. 15D, The miR-142-3p expression asdetermined by quantitative reverse transcription polymerase chainreaction analysis was down regulated during macrophage differentiationbut preferentially in the immunosuppressive M2 macrophages relative tothe proinflammatory M1 macrophages. Error bars represent standarddeviations. A paired two-sided Student t test was used. n=6, *P=0.03.

FIGS. 16A-D. miR-142-3p Interacts with the TGFβR1 Pathway. FIG. 16A,Western blot analysis of the predicted miR-142-3p targets as indicatedby bioinformatics tools in M1 and M2 macrophages untreated (blank),transfected with scramble control (ctrl), transfected with miR-142-3p(miR), or transfected with anti-miR-142-3p (anti). Similar result wasobserved in 3 replicates. FIG. 16B, miR-142-3p overexpression inhibitsthe downstream target, p-SMAD2, in M2 macrophages. After 5 days, the M1and M2 macrophages were treated with TGF-β1, transfected with scramblecontrol (ctrl) or miR-142-3p (miR) and then measured for p-SMAD2.Similar result was observed in 3 replicates. FIG. 16C, Sequence of thepredicted miR-142-3p binding site (SEQ ID NO:27) on the TGFBR1-3′UTR(upper; SEQ ID NO:28) and the mutated TGFBR1-3′UTR sequence whichpotentially disrupts miR-142-3p binding (lower; SEQ ID NO:29). FIG. 16D,The relative luciferase activity in HeLa cells after transfection withmiR-142-3p− in conjunction with the parental luciferase vector, the wildtype TGFBR1 3′-UTR or the miR-142-3p binding site mutant reporterconstruct. Luciferase activity is shown relative to the parentalluciferase vector and error bars represent standard deviations.

FIGS. 17A-B. miR-142-3p Transfection and TGFβR1 blockade InducesSelective Apoptosis in M2 Macrophages. (FIG. 17A) Representative flowanalysis cytometry and summarized data demonstrating more earlyapoptosis (Annexin V⁺7-AAD⁻) and late apoptosis/cell death (AnnexinV⁺7-AAD⁺) induced in M2- than in M1-committed monocytes at 48 hoursafter the miR-142-3p transfection (n=9). *P=0.02. (FIG. 17B)Representative flow analysis cytometry and summarized data set ofblockade of TGFβR1 by the antagonist SB431542 and LY364947 demonstratinginduced preferential M2 macrophage apoptosis (n=6). *P=0.04 and**P=0.001.

FIGS. 18A-D. miR-142-3p Inhibits In Vivo Glioma Growth. The treatmentschema (FIG. 18A) and volume of subcutaneous (s.c.) GL261 tumors (FIG.18B) in C57BL/6J mice treated intravenously with scramble control andmiR-142-3p starting on day 5 (n=5 per group). Linear mixed models werefit to assess tumor growth and F-test was used. Standard deviations areshown. n=5/group, *P=0.03. The in vivo experiment was duplicated withsimilar results. Treatment schema (FIG. 18C) and graph of theKaplan-Meier estimate of survival time in C57BL/6J mice implanted withintracerebral (i.c.) GL261 gliomas (FIG. 18D), which showed thatmiR-142-3p improved survival in the miR-142-3p-treated group relative tothe scramble controls. Log rank tests were used to compare overallsurvival between groups. n=10/group. *P=0.03.

FIGS. 19A-D: miR-142-3p Inhibits Glioma-infiltrating Macrophages.Treatment schema (FIG. 19A) and graph of the Kaplan-Meier estimate ofsurvival time (FIG. 19B) demonstrating improved survival inmiR-142-3p-treated Ntv-a mice transfected with the RCAS-PDGFB andRCAS-Bcl-2 transgenes compared with scramble control. Log rank testswere used to compare overall survival between groups. n=9/group.*P=0.03.FIG. 19C, Immunohistochemistry demonstrating staining with anti-F4/80antibodies to identify glioma-infiltrating macrophages. Left panel:representative images (max: ×400) of mice treated with scramble controland miR-142-3p respectively. Right panel: quantification ofglioma-infiltrating macrophage and comparison between the two groups.Scale bar=50 μm. FIG. 19D, The correlation between the percentage ofglioma-infiltrating F4/80+ macrophages and the survival duration ofmiR-142-3p treated mice. R2=0.303.

FIGS. 20A-C: (FIG. 20A) miR-142-3p expression was significantlyupregulated after transfection with a miR-142-3p precursor into the M1and M2 macrophages. (FIG. 20B) Transfection of miR-142-3p did not changethe expression levels of TGFβR1 mRNA as detected by RT-PCR. (FIG. 20C)Summarized TGF-β2 ELISA data from M1 and M2 macrophages demonstratingmiR-142-3p transfection does not alter secretion.

FIGS. 21A-C: (FIG. 21A) After the miR-142-3p precursor/inhibitortransfection, the expression of the general macrophage marker CD68 didnot significantly change in either the M1 or M2 macrophages (light greyis isotype control; black is CD68). (FIG. 21B) Expression of CD163, aspecific marker for M2 macrophages, was down regulated upon theoverexpression of miR-142-3p (light grey is isotype control; black isCD68). CD163 expression in M2 was upregulated by the miR-142-3pinhibitor (anti-miR). (FIG. 21C) Summarized data of the effects ofmiR-142-3p in macrophages. *P=0.02, n=8.

FIGS. 22A-D: (FIG. 22A) TGFβR1 was down regulated in both M1 and M2macrophages for two different TGFβR1 siRNAs (set #1 and set #2). (FIG.22B) Preferential apoptosis was observed in M2 macrophages compared toM1 macrophages in both siRNA treated group. (FIG. 22C) miR-142-3pexpression was significantly upregulated after transfection with amiR-142-3p precursor into the gCSCs. (FIG. 22D) Neither early apoptosis(Annexin V⁺7-AAD⁻) nor late apoptosis/cell death (Annexin V⁺7-AAD⁺) wasinduced in gCSCs transfected with miR-142-3p.

FIGS. 23A-B. FIG. 23A, The treatment schema of tumor-free C57BL/6J mice(n=5 per group) with scramble control or miR-142-3p duplex is shown.FIG. 23B, No significant change in the cell count was found forperipheral monocytes or T-cells in miR-142-3p treated mice relative tothe scramble control (P>0.05).

FIGS. 24A-D. FIG. 24A, Spleen macrophages from miR-142-3p treated tumoranimals presented a more M1-like differentiated phenotype. Bars indicatethe mean proportion (±SEM) of the percent of CD11b+ macrophagesproducing the cytokines Specifically, the percentage of CD11b+IL-6+M2macrophages was reduced to 51.1% in the miR-142-3p group relative to71.8% in the control group (P=0.013). Furthermore, the CD11b+ TNFα+M1macrophage percentage was increased from 13.9% in the control group to22.7% in the miR-142-3p group (P=0.032). FIG. 24B, Representative flowcytometric analysis of MHCII/33D1 surface expression on mature dendriticcells (mDCs) in splenocytes of GL261 tumor-bearing mice treated withmiR-142-3p or scramble control. Percentages represent the proportion ofthe MHCII+33D1+ cell subset. FIG. 24C, No significant changes ofeffector cytokine production in CD8+ T-cells were found betweenmiR-142-3p and scramble treated groups. D) CD8+ T-cells frommiR-142-3p-treated mice display similar cytotoxicity to that displayedby CD8+ T-cells from control mice E:T (effector T-cells: tumor cells)ratio 1, 2, 3 and 4 represent 1:1, 5:1, 10:1 and 20:1, respectively.Data are combined from 3 mice. P-value of statistical analysis (unpairedt-test) was shown above each bar graph.

FIGS. 25A-C: miR-138 binds the 3′ UTR of CTLA-4 and PD-1. FIG. 25A. Thethree predicted miR-138 binding sites in the 3′ un-translated region ofCTLA-4 are noted with their sequences. The mutational alterations arenoted for each luciferase expression construct. FIG. 25B. The miR-138PD-1 binding site sequence. FIG. 25C. A significant decrease inluciferase expression is seen when the cells are co-transfected with areporter plasmid containing the wild-type 3′ UTR of PD-1 and miR-138(WT-mir138 vs WT-scr, 18% decrease in relative luciferase expression,p<0.05); whereas this difference is abolished when the mutant 3′ UTRreporter plasmid is evaluated (Mut-scr vs Mut-mir138).

FIGS. 26A-C: miR-138 inhibits human checkpoint expression in Tregs.Healthy donor human CD4+ T cells were stimulated by anti-CD3/CD28 Absfor 48 h in the absence or presence of TGF-β to induce CTLA-4, PD-1 andFoxP3+ Tregs and subsequently transfected with miR-138 or scramblecontrol. MiR-138 down modulated the expression of PD-1 (FIG. 26A),CTLA-4 (FIG. 26B) and FoxP3 (FIG. 26C) in CD4 T cells. Solid grey filledhistogram is isotype control, dashed histogram is scramble control andsolid black line is miR-138. Representative histograms are shown asabove and summary data dot plots are shown below in which each dotrepresents the analysis of one human donor's peripheral CD4+ T cells(n=5). * indicates P<0.05.

FIGS. 27A-B: miR-138 exerts potent efficacy to suppress GL261 tumors ina syngeneic mouse model. FIG. 27A. The treatment schema and the volumesof subcutaneous GL261 tumors in C57BL/6J mice treated intravenously witheither miR-124 or scramble control, or left untreated starting on day 5(n=10 group/experiment). The figure is the result of a single experimentbut was repeated with identical results. In the miR-138 group, *denotesa P value of <0.01 compared with both the untreated and scramble controltumors. Standard deviations are shown. Arrows indicate days of treatmentand tumor size measurements. FIG. 27B. Treatment schema and graph of theKaplan-Meier estimate demonstrating survival of C57BL/6J mice withintracranial GL261 that were treated intravenously with miR-138 versusscramble control. miR-138 treatment resulted in a marked increase inmedian survival in comparison to scramble control (33.5 versus 23.5days; p=0.01).

FIGS. 28A-B: The therapeutic effect of miR-138 is immune mediated. FIG.28A. Treatment schema and graph of the Kaplan-Meier estimatedemonstrating the lack of therapeutic effect of miR-138 in nude micewith intracerebral GL261 gliomas (n=8 in scramble miRNA group, 7 inmiR-138 treatment group, P=0.87). FIG. 28B. Photomicrograph (400×)showing FoxP3 expressing lymphocytes within GL261 tumors treated withscramble control versus miR-138. The dot plot graph summarizes thenumber of FoxP3+ cells per 1000 between scramble and miR-138 treatedintracerebral GL261 tumors.

FIGS. 29A-C: Kaplan-Meier curves demonstrate that there is nosignificant difference in survival between high expression and lowexpression of miR-138 utilizing data from 383 glioblastoma patients inThe Cancer Genome Atlas comparing (FIG. 29A) the upper 50% versus lower50% level of expression or (FIG. 29B) the top 20% versus bottom 20%level of expression. FIG. 29C. The expression of miR-138 mRNA isdiminished in gCSCs and glioblastomas relative to normal brain byquantitative PCR.

FIGS. 30A-C: FIG. 30A. Kaplan-Meier survival estimates stratified by thepresence or absence of PD-L1 expression based on TCGA data sets. Therewas a significant decrease in survival with increased PD-L1 expression(p=0.0018, log-rank test). FIG. 30B. Representative microphotograph ofimmunohistochemical staining for PD-L1 on the glioblastoma TMA. FIG.30C. Histograms demonstrating the amount of PD-L1 expression relative toisotype controls in gCSCs and in glioma cell lines.

FIG. 31: Combination Therapies Involving miR-138 miR-142-3p, and/ormiR-138.

FIG. 32: Formulation equivalency studies of miRNA124 in nanoparticles.Graph of the Kaplan-Meier estimate demonstrating survival in theintracerebral GL261 intracerebral murine model treated with variousformulations of miR-124.

FIG. 33: Formulation equivalency studies and pharmacokinetics of immunemodulatory miRNAs. Non-tumor bearing C57BL/6J mice were administeredmiR-124+lipofectamine i.v. once and subsequently terminated at thedesignated time points. The liver, peripheral blood mononuclear cells(PBMCs) and serum were subsequently analyzed for miR-124 expression byquantitative PCR.

FIG. 34: p-STAT3 expression levels were decreased in the T-cells fromthe peripheral blood and gliomas in the miR-124 treated mice.Representative histograms were shown. The p-STAT3 expression levelsrelative to isotype control in the peripheral blood T-cells in themiR-124 treated mice (1.4±0.2%) was down-modulated compared to scrambletreated mice (17.9±1.9%) (P=0.007; n=3) and glioma-infiltrating T-cells(miR-124: 4.3±0.1%; scramble: 18.2±4.3%; P=0.007), but not within thesplenic T-cells (miR-124: 16.8±0.9%; scramble: 12.9±2.7%; P=0.07). Theisotype is light grey dashed lines, the scramble miRNA control is thedashed black line and miR-124 is the solid grey line.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Definitions

A nucleic acid will generally contain phosphodiester bonds, althoughnucleic acid analogs may be included that have at least one differentlinkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, orO-methylphosphoroamidite linkages and peptide nucleic acid backbones andlinkages. Other analog nucleic acids include those with positivebackbones; non-ionic backbones, and non-ribose backbones, includingthose described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which areincorporated by reference. Nucleic acids containing one or morenon-naturally occurring or modified nucleotides are also included withinone definition of nucleic acids. The modified nucleotide analog may belocated for example at the 5′-end and/or the 3′-end of the nucleic acidmolecule. Representative examples of nucleotide analogs may be selectedfrom sugar- or backbone-modified ribonucleotides. It should be noted,however, that also nucleobase-modified ribonucleotides, i.e.ribonucleotides, containing a non-naturally occurring nucleobase insteadof a naturally occurring nucleobase such as uridines or cytidinesmodified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromouridine; adenosines and guanosines modified at the 8-position, e.g.8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O- andN-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. The2′-OH-group may be replaced by a group selected from H, OR, R, halo, SH,SR, NH₂, NHR, NR₂ or CN, wherein R is C₁-C₆ alkyl, alkenyl or alkynyland halo is F, Cl, Br or I. Modified nucleotides also includenucleotides conjugated with cholesterol through, e.g., a hydroxyprolinollinkage as described in Krutzfeldt et al. (2005); Soutschek et al.(2004); and U.S. Patent Publication No. 20050107325, which areincorporated herein by reference. Modified nucleotides and nucleic acidsmay also include locked nucleic acids (LNA), as described in U.S. PatentPublication No. 2002/0115080, U.S. Pat. No. 6,268,490, and U.S. Pat. No.6,770,748, which are incorporated herein by reference. LNA nucleotidesinclude a modified extra methylene “bridge” connecting the 2′ oxygen and4′ carbon of the ribose ring. The bridge “locks” the ribose in the3′-endo (North) conformation, which is often found in the A-form of DNAor RNA. LNA nucleotides can be mixed with DNA or RNA bases in theoligonucleotide whenever desired. Such oligomers are commerciallyavailable from companies including Exiqon (Vedbaek, Denmark). Additionalmodified nucleotides and nucleic acids are described in U.S. PatentPublication No. 20050182005, which is incorporated herein by reference.Modifications of the ribose-phosphate backbone may be done for a varietyof reasons, e.g., to increase the stability and half-life of suchmolecules in physiological environments, to enhance diffusion acrosscell membranes, or as probes on a biochip. Mixtures of naturallyoccurring nucleic acids and analogs may be made; alternatively, mixturesof different nucleic acid analogs, and mixtures of naturally occurringnucleic acids and analogs may be made.

In some embodiments, a LNA or other nucleic acid analog may be producedvia methods involving use of an enzyme. Methods for producing LNAinclude the use of an enzyme or polymerase have been shown, e.g., inPinheiro et al. (2012). In some embodiments, a polymerase may be used inthe synthesis of C5-ethynyl locked nucleic acids, LNA, cyclohexenylnucleic acids (CeNA), anhydrohexital nucleic acids (HNA), orthreofuranosyl nucleid acids (TNA) (see, e.g., Veedu et al., 2010;Pinheiro et al., 2012). Producing LNA via the use of an enzyme maysignificantly reduce the costs associated with the production of LNA.

In certain embodiments, a LNA may be administered to a subject, such asa mammal, mouse, rat, dog, primate, or human subject. It is anticipatedthat a miRNA of the present invention (e.g., miR-124, miR-142, ormiR-138) does not need to be LNA. In various embodiments, a similareffect may be achieved using one or more unmodified miRNA sequenceseither alone, as a “naked miRNA,” or comprising one or more modification(e.g., to reduce in vivo degradation, improve pharmacokinetics, etc.).

In some embodiments, a modified nucleic acid that is not a LNA may beadministered to an individual to treat a cancer or hyperproliferativedisease. For example, an antisense nucleic acid comprising a 2′-4′conformationaly restricted nucleoside analogue may be used. Previouswork involving short oligonucleotides with a 2′-4′ conformationalyrestricted nucleoside analogues has shown that these molecules mayexhibit increased potency without increased toxicity in animals (Seth etal., 2009). Alternately, a cyclohexenyl nucleic acid (CeNA), ananhydrohexital nucleic acid (HNA), or a threofuranosyl nucleid acid(TNA) may be used as, or the modification may be included in, a miRNA ofthe present invention such as, e.g., miR-124, miR-142, or miR-138.

LNA (or 2′-4′ BNA) generally refers to a 2′-OMe nucleoside (A, below)where the methyl group is constrained back to the 4′-position of thefuranose ring system. The 2′-4′ constraint enforces an N-type sugarpucker of the furanose ring, which may result in improved hybridizationwith complementary RNA. By use of a similar strategy, constraining theethyl chain in the MOE residue back to the 4′-position of the furanosering system can be used to make nucleosides E (R-constrained MOE orR-cMOE) and F (S-cMOE) below (Seth et al., 2008). The methoxymethylgroups in cMOE nucleosides may mimic the steric and hydration attributesof MOE nucleosides and may, in some embodiments, improve the safetyprofile of antisense oligonucleoties containing these modifications.(Teplova et al., 1999).

“Promoter” as used herein may mean a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter may also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter may bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter may regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

II. MicroRNAs (miRNAs)

MicroRNAs (miRNAs) are short, non-coding RNAs that can target andsubstantially silence protein coding genes through 3′-UTR elements.Important roles for miRNAs in numerous biological processes have beenestablished, but comprehensive analyses of miRNA function in complexdiseases are lacking. MiRNAs are initially transcribed as primary miRNAs(pri-miRNAs) that are then cleaved by the nuclear RNAses Drosha andPasha to yield precursor-miRNAs (pre-miRNAs). These precursors arefurther processed by the cytoplasmic RNAse III dicer to form shortdouble stranded miR-miR* duplexes, one strand of which (miR) is thenintegrated into the RNA Induced Silencing Complex (RISC) that includesthe enzymes dicer and Argonaute (Ago). The mature miRNAs (˜17-24nt)direct RISC to specific target sites located within the 3′UTR of targetgenes. Once bound to target sites, miRNAs represses translation throughmRNA decay, translational inhibition and/or sequestration intoprocessing bodies (P-bodies) (Eulalio et al., 2008; Behm-Ansmant et al.,2006; Chu and Rana, 2006). Recent estimates find that over 60% ofprotein coding genes carry 3′-UTR miRNA target sites (Friedman et al.,2009). In this regard, miRNAs act as key regulators of processes asdiverse as early development (Reinhart et al., 2000), cell proliferationand cell death (Brennecke et al., 2003), apoptosis and fat metabolism(Xu et al., 2003), and cell differentiation (Chen, 2004; Dostie et al.,2003). In addition, studies of miRNA expression in chronic lymphocyticleukemia (Calin et al., 2008), colonic adenocarcinoma (Michael et al.,2003), Burkitt's lymphoma (Metzler et al., 2004), cardiac disease (Zhaoet al., 2007) and viral infection (Pfeffer et al., 2004) suggest vitallinks between miRNA and numerous diseases.

miRNAs thus far observed have been approximately 21-22 nucleotides inlength and they arise from longer precursors, which are transcribed fromnon-protein-encoding genes. See review of Carrington et al. (2003). Theprecursors form structures that fold back on each other inself-complementary regions; they are then processed by the nucleaseDicer in animals or DCL1 in plants. miRNA molecules interrupttranslation through precise or imprecise base-pairing with theirtargets. In some embodiments, a miRNA may be used therapeutically oradministered to a subject, such as a human patient, to treat a diseasesuch as, e.g., cancer; alternately, in some embodiments, a nucleic acidthat is complementary to the miRNA may be therapeutically administeredto a subject in vivo or used in vitro to generate the desiredtherapeutic miRNA (e.g., miRNA-142-3p, miRNA-142-3p, miRNA-124, ormiRNA-138). In this way, the complementary nucleic acid may be used as atemplate to generate the desired therapeutic miRNA (e.g., miRNA-142-3p,miRNA-142-3p, miRNA-124, or miRNA-138).

III. Adoptive Immunotherapies

In some embodiments, a miRNA of the present invention, such as miR-124,miR-142, or miR-138 may be used as an immunotherapeutic. Without wishingto be bound by any theory, the below examples indicate that these miRNAcan activate the immune systems response to a cancer. These miRNAs wereselected based on their ability to block tumor-mediated immunesuppression or immune checkpoints. A previous therapeutic limitation ofusing miRNAs for solid malignancies has been insufficient tumortargeting and penetration. This limitation may be overcome by activatingor exploiting the immune system to gain access to the tumor. In someembodiments, circulating immune cells may be among the first cells thatcontact an administrated miRNA. Without wishing to be bound by anytheory, in some embodiments, immune system responses may mediate theanti-tumor effect, thus in some embodiments the mode of administrationdoes not require miRNA to make direct contact with the tumor, e.g.,intratumoral injection is not required in some embodiments. Although amiRNA as disclosed herein may be directly administered (e.g.,intravenously, etc.) into a human subject, the miRNAs may also beutilized to modify a wide variety of pre-existing immunotherapeuticapproaches including but not exclusive to: adoptive T-cell transfers, NKimmunotherapy, dendritic cell immunotherapy, and/or a viralimmunotherapy to enhance their therapeutic effect. The miRNAs could beused to directly modify an adoptive immunotherapy or given concurrentlywith the adoptive immunotherapy. For example, isolated CD3+ T-cells maybe transfected with miR-124, and their numbers expanded in vitro beforeadoptively transferring these cells into patients. In mice models, thistype of miR-124 transfection in the CD3+ T cells was observed to inhibitp-STAT3 in the adoptively transferred T cells, and a significantenhancement of the anti-tumor therapeutic effects in comparison tounmodified CD3+ T cells was observed. As shown in the below examples,miR-124 transfected adoptively transferred T cells can induce potentanti-tumor immune effector responses while minimizing Tregs in the tumormicroenvironment. In various embodiments, another miRNA of the presentinvention, such as, e.g., miR-142 or miR-138, may be substituted for orused in combination with miR-124, as described above.

IV. Clinical Information A. Definitions

“Treatment” and “treating” as used herein refer to administration orapplication of a therapeutic agent to a subject or performance of aprocedure or modality on a subject for the purpose of obtaining atherapeutic benefit of a disease or health-related condition.

The term “therapeutic benefit” or “therapeutically effective” as usedthroughout this application refers to anything that promotes or enhancesthe well-being of the subject with respect to the medical treatment ofthis condition. This includes, but is not limited to, a reduction in thefrequency or severity of the signs or symptoms of a disease.

“Prevention” and “preventing” are used according to their ordinary andplain meaning to mean “acting before” or such an act. In the context ofa particular disease or health-related condition, those terms refer toadministration or application of an agent, drug, or remedy to a subjector performance of a procedure or modality on a subject for the purposeof blocking the onset of a disease or health-related condition.

The term “compound” refers to any chemical entity, pharmaceutical, drug,and the like that can be used to treat or prevent a disease, illness,sickness, or disorder of bodily function. Compounds comprise both knownand potential therapeutic compounds. A compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. A “known therapeutic compound” refers to a therapeuticcompound that has been shown (e.g., through animal trials or priorexperience with administration to humans) to be effective in suchtreatment. In other words, a known therapeutic compound is not limitedto a compound efficacious in the treatment of asthma.

A “sample” is any biological material obtained from an individual. Forexample, a “sample” may be a blood sample or a lung tissue sample.

B. Dosage

A pharmaceutically effective amount of a therapeutic agent as set forthherein is determined based on the intended goal, for example inhibitionof cell death. The quantity to be administered, both according to numberof treatments and dose, depends on the subject to be treated, the stateof the subject, the protection desired, and the route of administration.Precise amounts of the therapeutic agent also depend on the judgment ofthe practitioner and are peculiar to each individual.

For example, a dose of the therapeutic agent may be about 0.0001milligrams to about 1.0 milligrams, or about 0.001 milligrams to about0.1 milligrams, or about 0.1 milligrams to about 1.0 milligrams, or evenabout 10 milligrams per dose or so. Multiple doses can also beadministered. In some embodiments, a dose is at least about 0.0001milligrams. In further embodiments, a dose is at least about 0.001milligrams. In still further embodiments, a dose is at least 0.01milligrams. In still further embodiments, a dose is at least about 0.1milligrams. In more particular embodiments, a dose may be at least 1.0milligrams. In even more particular embodiments, a dose may be at least10 milligrams. In further embodiments, a dose is at least 100 milligramsor higher.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above. Dosages of nucleic acid or LNA which may beused include, for example, about from 10-100 mg (LNA or nucleic acid)/gbody weight, about 25-75 mg (LNA or nucleic acid)/g body weight, aboutmg (LNA or nucleic acid)/g body weight, or any range derivable therein.A dosage of about 50 mg (LNA or nucleic acid)/g mouse body weight wasobserved to be effective to substantially inhibit allergic orinflammatory lung responses in mice in vivo. In some embodiments, a doseof about 0.01-5 mg/kg, about 0.01-1 mg/kg, or about 0.03-1 mg/kg may beadministered to a subject, such as a human patient.

The dose can be repeated as needed as determined by those of ordinaryskill in the art. Thus, in some embodiments of the methods set forthherein, a single dose is contemplated. In other embodiments, two or moredoses are contemplated. Where more than one dose is administered to asubject, the time interval between doses can be any time interval asdetermined by those of ordinary skill in the art. For example, the timeinterval between doses may be about 1 hour to about 2 hours, about 2hours to about 6 hours, about 6 hours to about 10 hours, about 10 hoursto about 24 hours, about 1 day to about 2 days, about 1 week to about 2weeks, or longer, or any time interval derivable within any of theserecited ranges.

In certain embodiments, it may be desirable to provide a continuoussupply of a pharmaceutical composition to the patient. This could beaccomplished by catheterization, followed by continuous administrationof the therapeutic agent. The administration could be intra-operative orpost-operative.

V. Pharmaceutical Compositions and Routes for Administration to Patients

Some embodiments of the present invention involve administration ofpharmaceutical compositions. Where clinical applications arecontemplated, pharmaceutical compositions will be prepared in a formappropriate for the intended application. Generally, this will involvepreparing compositions that are essentially free of pyrogens, as well asother impurities that could be harmful to humans or animals.

In some embodiments, a miRNA may be delivered in a lipid emulsion, aliposome, a nanoparticle, an exosome, or in a viral vector. The liposomemay be a unilamellar, multilamellar, or multivesicular liposome. It isanticipated that a wide variety of liposomes and exosomes may be usedwith the present invention. For example, in some embodiments, a siliconenanoparticle may be used to deliver a miRNA to a cell (e.g., asdescribed in Bharali et al. PNAS (2005) 102(32): 11539-11544, which isincorporated by reference in its entirety). Liposomes may compriseN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP) or Lipofectamine™. In some embodiments, a delivery systeminvolving chitosan may be used as described, e.g., in Lu et al. (CancerCell (2010) 18:185-197), which is incorporated by reference in itsentirety without disclaimer. In some embodiments, a nanovector may beused to deliver a miRNA to a subject; nanovectors are described, e.g.,in Pramanik et al. Mol Cancer Ther (2011) 10:1470-1480, which isincorporated by reference in its entirety without disclaimer.

One will generally desire to employ appropriate salts and buffers inpreparing compositions of therapeutic agents. Buffers also will beemployed when recombinant cells are introduced into a patient. Aqueouscompositions of the present invention comprise an effective amount ofthe therapeutic agent, dissolved or dispersed in a pharmaceuticallyacceptable carrier or aqueous medium. The phrases “pharmaceuticallyacceptable” or “pharmacologically acceptable” refers to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes solvents,buffers, solutions, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the likeacceptable for use in formulating pharmaceuticals, such aspharmaceuticals suitable for administration to humans. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active ingredients of the present invention, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions, providedthey do not inactivate the therapeutic agents of the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue is available via that route. Administration may beby any method known to those of ordinary skill in the art, such asintravenous, intradermal, subcutaneous, intramuscular, intraperitoneal,intrathecal, intralymphatic, inhalation, intranasal, or by directinjection into the tumor. Other modes of administration include oral,buccal, and nasogastric administration. The active compounds may also beadministered parenterally or intraperitoneally. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions, asdescribed supra. In particular embodiments, the composition isadministered to a subject using a drug delivery device. For example, thedrug delivery device may be a catheter or syringe. Alternatively, themiRNAs could be incorporated into a polymer or polifeprosan andimplanted into a resection cavity or delivered by convection enhanceddelivery.

By way of illustration, solutions of the active compounds as free baseor pharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils and by spray-dry techniques. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying, freeze-drying andspray-drying techniques which yield a powder of the active ingredient(s)plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In preferred embodiments systemic formulations of the miRNA arecontemplated. Systemic formulations include those designed foradministration by injection, e.g. subcutaneous, intravenous,intramuscular, intrathecal or intraperitoneal injection, as well asthose designed for transdermal, transmucosal, inhalation, oral orpulmonary administration. In some embodiments, a miRNA of the presentinvention is delivered by intravenous or intratumoral injection. Inembodiments, the mode of administration is not intratumoral injection.

For injection, the proteins of the embodiments may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks' solution, Ringer's solution, or physiological saline buffer.The solution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

For oral administration the therapeutic agents of the present inventiongenerally may be incorporated with excipients. Any excipient known tothose of ordinary skill in the art is contemplated. In some embodiments,administration is not oral. In some embodiments, it may be advantageousto modify any miRNA that will be administered orally to reducedegradation. In some embodiments it may be preferable to administer amiRNA via a route that is not oral, such as, e.g., an intravenous,parenteral, intraperitoneal, intratumoral, or subcutaneous, etc. route.In some embodiments, a miRNA of the present invention is administeredintravenously.

The compositions of the present invention generally may be formulated ina neutral or salt form. Pharmaceutically-acceptable salts include, forexample, acid addition salts (formed with the free amino groups of theprotein) derived from inorganic acids (e.g., hydrochloric or phosphoricacids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic,and the like). Salts formed with the free carboxyl groups of the proteincan also be derived from inorganic bases (e.g., sodium, potassium,ammonium, calcium, or ferric hydroxides) or from organic bases (e.g.,isopropylamine, trimethylamine, histidine, procaine and the like).

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous and intraperitonealadministration. Preferably, sterile aqueous media are employed as isknown to those of skill in the art, particularly in light of the presentdisclosure. By way of illustration, a single dose may be dissolved in 1ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15^(th) Edition,pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

VI. Cancers

In some aspects, a miRNA of the present invention may be administered toa subject or individual to treat a cancer. In some embodiments, thecancer is a brain cancer, a glioma, a neuroblastoma, glioblastoma,glioblastoma multiforme, an oligodendroglioma or metastatic tumor to thebrain. In various aspects, it is anticipated that miRNAs of the presentinvention may be used to treat virtually any malignancy. In someembodiments, the cancer may suppress the immune system of the subject orindividual with the cancer. In some embodiments and as shown in thebelow examples, miRNA as provided herein can suppress or reversecancer-mediated immune suppression and allow for immune recognition andclearance of the malignancy.

In some embodiments, a miRNA of the present invention may be used topromote or enhance clearance or attack of a cancer by the immune systemof a subject or individual with the cancer. Cancer cells that may betreated with miRNA according to the embodiments include but are notlimited to cells from the bladder, blood, bone, bone marrow, brain,breast, colon, esophagus, gastrointestine, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas,testis, tongue, cervix, or uterus. In addition, the cancer mayspecifically be of the following histological type, though it is notlimited to these: neoplasm, malignant; carcinoma; carcinoma,undifferentiated; giant and spindle cell carcinoma; small cellcarcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell tumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignantlymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse;malignant lymphoma, follicular; mycosis fungoides; other specifiednon-hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mastcell sarcoma; immunoproliferative small intestinal disease; leukemia;lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcomacell leukemia; myeloid leukemia; basophilic leukemia; eosinophilicleukemia; monocytic leukemia; mast cell leukemia; megakaryoblasticleukemia; myeloid sarcoma; and hairy cell leukemia. In certain aspects,the tumor may comprise an osteosarcoma, angiosarcoma, rhabdosarcoma,leiomyosarcoma, Ewing sarcoma, glioblastoma, neuroblastoma, or leukemia.

VII. Combined Therapy

In another embodiment, it is envisioned to use a miRNA or a miRNAinhibitor as set forth herein in combination with other therapeuticmodalities. Thus, in addition to the therapies described above, one mayalso provide to the patient a more “standard” pharmaceutical therapy.Examples of other therapies include, e.g., a chemotherapy, aradiotherapy or radiation therapy, a cytokine therapy, a gene therapy,an immunotherapy, and/or a surgery. In some embodiments, a miRNA of thepresent invention such as, e.g., miR-124, miR-142, or miR-138 isadministered to a subject in combination with an adoptive immunotherapy.

The other therapeutic modality may be administered before, concurrentlywith, or following administration of the miRNA. The therapy using miRNAmay precede or follow administration of the other agent(s) by intervalsranging from minutes to weeks. In embodiments where the other agent andthe miRNA are administered separately, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that each agent would still be able to exert anadvantageously combined effect. In such instances, it is contemplatedthat one would typically administer the miRNA and the other therapeuticagent within about 12-24 hours of each other and, more preferably,within about 6-12 hours of each other, with a delay time of only about12 hours being most preferred. In some situations, it may be desirableto extend the time period for treatment significantly, however, whereseveral days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of a miRNA, orthe other agent will be desired. In this regard, various combinationsmay be employed. By way of illustration, where the miRNA is “A” and theother agent is “B”, the following permutations based on 3 and 4 totaladministrations are exemplary:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B

Other combinations are likewise contemplated. Non-limiting examples ofpharmacological agents that may be used in the present invention includeany pharmacological agent known to be of benefit in the treatment of acancer or hyperproliferative disorder or disease.

VIII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 miR-124 Systemically Enhances T Cell Mediated Anti GliomaImmune Clearance by Inhibiting STAT3 Signaling

A. Materials and Methods

1. miR Comparison of Glioblastoma to Normal Brain Tissue

This study was approved by the institutional review board at M.D.Anderson and conducted according to protocol #LAB03-0687. Tumors werepathologically confirmed as glioblastoma (World Health Organizationgrade IV) by a board-certified neuropathologist. Tumors were washed inRPMI1640 medium and dissected to remove blood products and surroundingnon-tumor brain tissue. The total tissue was broken down into smallerpieces and digested in digesting buffer from the cancer cell isolationkit (Panomics, Santa Clara, Calif.) for 2 hours. The cells weresuspended in RNAlater solution (Ambion, Austin, Tex.) in Rnase-freetubes and stored at 4° C. overnight; after 24 hours, they weretransferred to −80° C. until needed for total RNA extraction. Extractionwas performed using the mirVana kit (Ambion). Once extracted, RNA levelswere analyzed for concentrations and purity using UV/Vis spectroscopy at230, 260, and 280 nm.

Total RNA extracted from patients was sent to Phalanx Biotech Group(Belmont, Calif.) for microRNA and mRNA-gene expression analyses. TotalRNA from normal brain tissues was obtained from Biochain (Hayward,Calif.). The results of the glioblastoma human miRNA OneArray Microarrayv2 analysis were used to determine which miRs had significantdifferences in expression compared with normal donor miRs. Expressionaldifferences in terms of multiples (-fold differences) were calculatedwith Microsoft Excel, and miRs with the most significant differences inexpression levels were chosen for the miR target analysis usingTargetScan (Release 5.1)(Friedman et al., 2009). miRs of interest wereselected on the basis of putative targets and the degree of deviationfrom normal brain.

2. Real-Time PCR to Confirm Relative miRNA Expression Levels

Total RNA extracted from glioblastoma cells or gCSCs was used as thetemplate for reverse transcription using the TaqMan reversetranscription kit (Applied Biosystems, Carlsbad, Calif.) in athermocycler, per the manufacturer's instructions. Primers for reversetranscription and PCR were purchased for human miR-124, miR-21, U6 andU18 snRNAs (Applied Biosystems). U6 and U18 was used as an endogenouscontrol. cDNA was used as the template for real-time PCR. U18 andmiR-124 amplifications were run in triplicate using the TaqMan real-timePCR kit (Applied Biosystems) in the 7500 real-time PCR system (AppliedBiosystems). Further reactions, substituting water for the cDNAtemplate, were used as additional controls. Excel was used to calculatethe mean levels of each miR and the U18 internal control. The relativeexpression levels of miR-124 were compared with those of the internalcontrols, and a bar graph was generated.

3. Glioma Tissue Microarray and In Situ Hybridization

This study was conducted according to LAB09-0463, which was approved bythe institutional review board at MD Anderson and includes 235 patientswith different glioma grades. The TMAs consisted of resected gliomatissues from glioblastoma (n=150), gliosarcoma (n=6), anaplasticastrocytoma (n=24), anaplastic mixed oligoastrocytoma (n=9), anaplasticoligodendroglioma (n=16), mixed oligoastrocytoma (n=5),oligodendroglioma (n=24), low-grade astrocytoma (n=1), subependymoma(n=2), and normal brain (cortex; n=19), and they have been previouslydescribed (Barnett et al., 2007). For TMA construction, two 1-mm coreswere obtained per tumor sample. The time from resection to fixation wasless than 20 minutes in all cases, in accordance with the ClinicalLaboratory Improvement Amendments standard.

In situ hybridization was performed using the protocol developed byNuovo et al (2009) with some minor adjustments. Digoxigenin-labeled,locked nucleic acid-modified probes for miR-124 (hsa-miR-124) and thepositive control (U6, hsa/mmu/rno) were purchased from Exiqon (Vedbek,Denmark). In brief, 4-μm sections of the TMA blocks were placed in aheater at 59° C. overnight to attach cores to the silane-coated slide.Sections were deparaffinized with xylene (2×5 minutes), rehydrated withethanol (100%, 50%, & 25% for 5 minutes each), and treated withdiethylpyrocarbonate-treated water for 1 minute. Protease treatment wasperformed with pepsin solution (1.3 mg/ml) (Dako, Glostrup, Denmark) at37° C. for 50 minutes. After a post-fixation step in 4%paraformaldehyde, hybridization of the locked nucleic acid probe wascarried out in a Hybrite (Abbott Laboratories, Abbott Park, Ill.) at 60°C. for 5 minutes followed by 37° C. overnight (12-18 hours). Alow-stringency post-hybridization wash was performed at 4° C. instandard sodium citrate containing 2% bovine serum albumin for 5minutes, followed by incubation with anti-digoxigenin/alkaline phosphateconjugate antibodies (Enzo Diagnostics, Farmingdale, N.Y.) in a heaterat 37° C. for 30 minutes. The blue color was developed by incubating theslide with nitroblue tetrazolium and bromchloroindolyl phosphate (EnzoDiagnostics) at 37° C. The colorimetric reaction was monitored visuallyand stopped by placing the slides in water when background coloringstarted to appear. The TMA was analyzed by the study neuropathologist.In the assessment of miR-124 expression in gliomas, intervening neuronsin the infiltrating component were not considered positive.

4. miR-124 Transfection in gCSCs, Astrocytes and T-Cells

The precursor form of miR-124 (30 nM) and the scramble negative controlwere used to transfect gCSCs and T-cells using the siPORT NeoFXtransfection agent (Applied Biosystems) or Nucleofector transfection kit(Lonza, Allendale, N.J.). Cells were incubated for 72 hours at 37° C. todetermine cell surface marker expression and collect secreted cytokines.miR-124 expression was verified via RT-PCR after transfection. Themorphologic characteristics of the gCSCs were documented at 48 hoursafter the transfection. A rescue experiment of miR-124 inhibition wasaccomplished by cotransfection with a plasmid expressing wild-type,constitutively active STAT3 without a miR-124 binding 3′ UTR site(provided by Dr. Jinbo Yang).

5. In Vivo Experiments

The miR-124 duplex that mimics pre-miR-124a (sense:5′-UAAGGCACGCGGUGAAUGCCA-3′ (SEQ ID NO:4), antisense:3′-UAAUUCCGUGCGCCACUUACG-5′, (SEQ ID NO:5)) and the scramble controlmiRNA duplex (sense: 5′-AGUACUGCUUACGAUACGGTT-3′ (SEQ ID NO:6),antisense: 3′-TTUCAUGACGAAUGCUAUGCC-5′ (SEQ ID NO:7)) were synthesized(SynGen, San Carlos, Calif.). The sequence of murine miR-124 isidentical to human miR-124 on the basis of NCBI blast data. Thetreatment cohorts consisted of 20 μg of the miR-124 duplex or scramblecontrol in 10 of PBS mixed with the vehicle (80 μL PBS containing 10 μLlipofectamine 2000; Invitrogen) or the vehicle control (90 μl PBS+10 μllipofectamine 2000). The dosing was identical for intratumoral deliveryor intravenous infusion. Mice were maintained in the M.D. AndersonIsolation Facility in accordance with Laboratory Animal ResourcesCommission standards and handled according to the approved protocol08-06-11831.

6. Syngeneic Subcutaneous Model

The murine glioma GL261 cell line was obtained from the National CancerInstitute-Frederick Cancer Research Tumor Repository. On the basis ofPCR expression, miR-124 expression in GL261 cells was 350-fold less thanin normal murine brain. These cells were cultured in an atmosphere of 5%CO₂ and 95% humidified air at 37° C. in Dulbecco's modified Eagle'smedium (Life Technologies; Grand Island, N.Y.), supplemented with 10%fetal bovine serum (Atlanta Biologicals, Norcross, Ga.), 1%penicillin/streptomycin (Life Technologies, Grand Island, N.Y.), and 1%L-glutamine (Life Technologies). The GL261 glioma cell cultures weredivided every 3 days to ensure logarithmic growth. To inducesubcutaneous tumors, logarithmically growing GL261 cells were injectedinto the right hind flanks of 6-week-old C57BL/6J female mice or nudemice at a dose of 4×10⁵ cells suspended in 100 μl of Matrigel basementmembrane matrix (BD Biosciences). When palpable tumors formed(approximately 0.5 cm in diameter), the mice (n=10/group) were treatedby local tumor injection or intravenous injection. Tumors were measuredevery other day. Mice that showed signs of morbidity, high tumor burden,or skin necrosis were immediately euthanized according to M.D. Andersonguidelines. Tumor volume was calculated with slide calipers using thefollowing formula: V=(L×W×H)/2, where V is volume (mm³), L is the longdiameter, W is the short diameter, and H is the height. Likewise,miR-124 or scramble miR was delivered systemically via the tail veinevery other day.

7. Syngeneic Intracranial Glioma Model

To induce intracerebral tumors in C57BL/6J mice, GL261 cells werecollected in logarithmic growth phase, washed twice with PBS, mixed withan equal volume of 10% methyl cellulose in Improved modified Eagle'sZinc Option medium, and loaded into a 250-μl syringe (Hamilton, Reno,Nev.) with an attached 25-gauge needle. The needle was positioned 2 mmto the right of bregma and 4 mm below the surface of the skull at thecoronal suture using a stereotactic frame (Kopf Instruments, Tujunga,Calif.), as previously described (Heimberger et al., 2003). Theintracerebral tumorigenic dose for GL261 cells was 5×10⁴ in a totalvolume of 5 μl. Mice were then randomly assigned to control andtreatment groups (n=10/group). Animals were observed three times perweek, and when they showed signs of neurological deficit (lethargy,failure to ambulate, lack of feeding, or loss of >20% body weight), theywere compassionately killed. These symptoms typically occurred within 48hours of prior to death. The brains were removed and placed in 4%paraformaldehyde and embedded in paraffin.

8. Genetically Engineered Murine Models

Vector Constructs.

RCAS-PDGFB generation has been previously described (Dai et al., 2001).RCAS-STAT3 was created by amplifying the sequence encoding the cDNA byPCR using specially designed primers to enable directional cloning intoa Gateway entry vector. The proprietary Gateway LR recombinationreaction between the entry vector containing STAT3 and aGateway-compatible RCAS destination vector resulted in the RCAS-STAT3vector, which was sequence verified.

DF-1 Cell Transfection.

DF-1 immortalized chicken fibroblasts were grown in Dulbecco's modifiedEagle's medium with 10% fetal bovine serum in a humidified atmosphere of95% air/5% CO₂ at 37° C. Live virus was produced by transfecting plasmidversions of RCAS vectors into DF-1 cells using FuGene6. These cells werereplicated in culture.

Verification of STAT3 Expression in Vector.

Untransfected DF-1 cells were grown in culture, transfected withRCAS-STAT3, and allowed to replicate for two to three passages. Cellswere fixed with 4% paraformaldehyde, and immunocytochemical labeling wasperformed using standard methods. A rabbit polyclonal antibody againstSTAT3 (1:100; Cell Signaling Technology, Beverly, Mass.) and goatanti-rabbit Alexa Fluor 594 fluorescent conjugate (1:500; MolecularProbes, Carlsbad, Calif.) were used for detection. Prolong Goldanti-fade reagent with 4′-6-diamidino-2-phenylindole (DAPI) was used forlabeling cell nuclei. Staining was visualized with a Zeiss Axioskop 40microscope. Expression was secondarily validated by Western blotanalysis.

In vivo somatic cell transfer in transgenic mice. The transgenic Ntv-amice are mixtures of different strains, including C57BL/6, BALB/c,FVB/N, and CD1. To transfer genes via RCAS vectors, DF-1 producer cellstransfected with a particular RCAS vector (5×10⁴ DF-1 cells in 1-2 μl ofPBS) were injected into the frontal lobes of Ntv-a mice at the coronalsuture of the skull using a Hamilton Gastight syringe. The mice wereinjected on postnatal days 1 or 2, when the number of Nestin+ cellsproducing TVA is the highest. The mice were killed 90 days afterinjection or sooner if they demonstrated morbidity related to tumorburden. Their brains were removed and analyzed for tumor formation.Histologic verification of tumor formation and determination of tumorgrade were performed by a neuropathologist.

Animal Randomization.

Twenty-one days after introducing the glioma-inducing transgenesRCAS-PDGFB and RCAS-STAT3, littermates to the treatment or control group(n=9/group) were randomly assigned. Mice were treated intravenously onMonday, Wednesday, and Friday for 3 weeks. After 90 days, the animalswere compassionately killed, the CNS was fixed, and the tumors wereanalyzed immunohistochemically.

9. Statistical Analysis

The distribution of each continuous variable was summarized by its mean,standard deviation, and range. The distribution of each categoricalvariable was summarized in terms of its frequencies and percentages.Continuous variables were compared between treatment groups by atwo-sample t test. In the case of comparing two paired groups, a pairedt test is conducted. Kaplan-Meier curves were used to estimateunadjusted time to event variables. Log-rank tests were used to compareeach time-to-event variable between groups. P values of less than 0.05(two-sided) were considered statistically significant. All statisticalanalyses were performed using the Statistical Package for the SocialSciences v.12.0.0 (SPSS, Chicago, Ill.) and SAS v. 9.1 (SAS Institute,Cary, N.C.). Error bars represent SD.

B. Results

1. miR-124 Expression in Gliomas

To determine the pattern of miR expression in glioblastoma relative tonormal brain tissue, the Human miRNA OneArray Microarray v2 was used.miR-124 emerged as a leading candidate, with a mean 24.6-fold decreasein expression from that seen in normal brain tissue (Table 1). Asubsequent analysis using reverse transcription-polymerase chainreaction (RT-PCR) confirmed that miR-124 was down regulated inglioblastoma GBM specimens (n=4), glioma cell lines (n=2), and gCSCs(n=4) compared with normal brain tissues (n=3) (FIG. 1A). When the gCSCswere placed under neural differentiation conditions, miR-124 expressionlevels were increased (FIG. 8). Because miR-124 was a leading candidatedown regulated in glioblastoma, as observed by ourselves and others (27,31), it determined whether it was decreased in other types of gliomas.Using a glioma tissue microarray and in situ hybridization, it was foundthat all glioma grades and types lacked miR-124 expression (FIG. 1B andTable 2). All cortex-containing neurons demonstrated positive expressionof miR-124 (n=19). No differences in survival time among glioblastomapatients were found on the basis of the relative but negligibleexpression of miR-124 in The Cancer Genome Atlas data set.

TABLE 1 Altered miRNA expression of glioblastoma relative to normalbrain Relative Relative miRNA downregulation miRNA upregulation miR-12424.6 miR-1273 3.7 miR-3172 13.8 miR-559 3.6 miR-138 13.4 miR-4286 3.2miR-3196 8.5 miR-3152 3.1 let-7b 7.3 miR-766 3 let-7e 6.9 miR-542-3p 2.7miR-1826 5.9 miR-1302 2.7 miR-1228* 5.8 miR-2355 2.7 miR-4284 5.6miR-1285 2.6 let-7d 5.6 miR-548c-5p 2.5 miR-3162 5.4 miR-1281 2.3miR-874 5.2 miR-1248 2.2 let-7c 5.2 miR-1272 2.1 miR-103 5 miR-488* 2.1miR-128 4.9 miR-3192 2.1 let-7a 4.7 miR-548d-5p 2.1 miR-26a 4.5 miR-31972.1 miR-762 4.5 miR-4323 2.1 miR-7 4.2 miR-3146 2

TABLE 2 miRNA expression incidence in gliomas % with miR-124 Tumorpathologic type WHO grade expression N Glioblastoma IV 0 150 GliosarcomaIV 0 6 Anaplastic astrocytoma III 0 24 Anaplastic mixedoligodendroglioma III 0 9 Anaplastic oligodendroglioma III 0 16 Mixedoligoastrocytoma II 0 5 Oligodendroglioma II 0 24 Low-grade astrocytomaII 0 1 Subependymoma I 0 2

2. miR-124 Interacts with the STAT3 Pathway

To determine which miRs interact with STAT3, TargetScan was used toidentify a group of miRs with conserved target sites in the STAT33′-UTR. Theoretically, these miRs can inhibit STAT3 expression and thusdown regulate STAT3-mediated immune suppression in glioblastoma. Thetop-rated candidates were miR-124, miR-17, miR-125, and miR-129, withaggregate P_(CT) scores of 0.85, 0.85, 0.84, and 0.58 (respectively)(Table 3).

TABLE 3 Predicted binding affinity of glioblastoma down-regulated miRNAsfor STAT3 Poorly Conserved sites conserved sites miRNA Total 8mer 7merTotal 8mer 7mer Aggregate P_(CT) miR-124 1 1 0 0 0 0 0.85 miR-17-5p 2 02 1 0 1 0.85 miR-125 1 1 0 0 0 0 0.84 miR-129 0 0 0 3 1 2 0.58 miR-29abc0 0 0 1 0 1 0.2 miR-150 0 0 0 1 0 1 0.2 miR-34a 0 0 0 1 0 1 0.15 miR-4250 0 0 1 0 1 0.14 miR-21 0 0 0 2 0 2 0.13 miR-221 0 0 0 1 0 1 0.12miR-383 0 0 0 1 0 1 0.11 miR-214 0 0 0 1 0 1 0.11

An additional analysis suggests that miR-124 is predicted to bind toSTAT3. Therefore, on the basis of these cumulative bioinformatics data,a mechanistic and therapeutic evaluation of miR-124 was performed.Because the predicted binding sites of miR-124 to STAT3 are in aconserved, homologous region (FIG. 1C), it determined whether miR-124directly inhibits STAT3 protein expression by binding to the 3′-UTR.miR-124-negative HeLa cells were transfected with pre-miR-124 plasmid orpre-miR-control plasmid. The 3′-UTR reporter activities of STAT3 wereassessed by luciferase assays. miR-124 inhibited STAT3 luciferaseactivity in cotransfected HeLa cells (FIG. 1D), whereas directedmutational alteration of the miR-124 3′-UTR STAT3 binding site (FIG. 1C)resulted in complete abolishment of miR-124 inhibition of luciferaseactivity in cotransfected HeLa cells (FIG. 1D). Subsequently, both STAT3and pSTAT3 expression at the protein level were inhibited by miR-124within gCSCs as detected by Western blot (FIG. 1E).

In addition to STAT3, TargetScan and other online software also suggestthat miR-124 may target other components of the STAT3 signaling pathwaysincluding IL6Rα, Tyk2, Src homology 2 domain-containing transformingprotein 1 (Shc1) and MAPK1 (Table 4). In order to test whether miR-124suppresses these predicted targets, the effect of forced expression ofmiR-124 in five gCSCs isolated from different glioblastoma patients(FIG. 1E) was investigated. Shc1 is also a preferred target of miR-124in gCSCs and is down regulated in all gCSCs treated with miR-124.pMAPK1/3 is down modulated in one gCSC cell line but this line isnotable for the lack of IL-6Rα expression, suggesting that p-STAT3activation may be due to alternative EGFR/MAPK1/3 dominant signaling.Although IL-6Rα, Tyk2, and MAPK1/3 are not preferred targets of miR-124in gCSCs by Western blot, miR-124 can target at least two key componentsin the STAT3 signaling pathway (FIG. 9). To determine if miR-124 candown modulate targets downstream of p-STAT3 such as miR-21, miR-21 levelin miR-124 overexpressing gCSCs by RT-PCR was measured and found thatmiR-21 expression was inhibited by miR-124 (FIG. 10).

TABLE 4 Target Scan predicted miR-124 binding sites of the moleculeswithin the STAT3 signaling pathway Total Conserved Sites PoorlyConserved Sites context Aggregate Total 8mer 7mer-m8 7mer-1A Total 8mer7mer-m8 7mer-1A Score Pct STAT3 1 1 −0.04 0.92 IL-6Rα 1 1 1 1 −0.3 0.8Tyk2 1 1 −0.33 <0.1 Shc1 1 1 −0.19 0.87 MAPK1 1 1 −0.06 0.95

3. miR-124 Reverses gCSC-Mediated Immune Suppression

To determine the phenotypic consequences of up regulating miR-124, humanimmune-suppressive gCSCs (13) were transiently transfected withprecursor miRs and confirmed the up regulation of miR-124 by RT-PCR. ThemiR-124 expression was increased in the range of 5-20,000 fold amongdifferent gCSCs. After 24 hours, gCSCs demonstrated increased adherenceto the bottom of the plate, which was more pronounced after 48 hours.Specifically, the typical neurosphere morphology of the gCSCs wasaltered to become petri dish-attached with an elongated configurationand with contact inhibition (FIG. 2A). In contrast, transfection ofastrocytes with miR-124 did not alter morphology, proliferation,apoptosis or cell cycle status. To characterize their immunologicalphenotype, gCSCs were assessed for their expression of majorhistocompatibility complex (MHC) I, MHC II, CD40, CD80, CD86, and B7-H1,by RT-PCR and flow cytometry after transfection with miR-124. No changeswere found in MHC I, MHC II, CD40, CD80, B7-H1 or CD86 mRNA and proteinexpression levels. To determine what immune-suppressive soluble factorsare affected by miR-124, the conditioned medium of miR-124- or scramble(control)-transfected gCSCs were analyzed using enzyme-linkedimmunosorbent assays (ELISAs) and cytokine and chemokine arrays. Here,lower levels of IL-8 (scramble: 5844±1108 pg/ml versus miR-124: 2115±672pg/ml; n=4, P<0.05), galectin-3 (scramble: 933±214 pg/ml versus miR-124:555±72 pg/ml; n=4, P<0.01), and MIC-1 (scramble: 13±4 pg/ml versusmiR-124: 4±1 pg/ml; n=4, P<0.05) (FIG. 8) but not of VEGF were found.Cytokine and chemokine array data revealed a modest decrease in levelsof TGF-β₂, macrophage migration inhibitory factor, Serpin E1, CX3CL1,CXCL10, CXCL16, and chemokine C-C motif-2, when miR-124 wasoverexpressed in gCSCs, but these findings were not statisticallysignificant.

To determine whether miR-124 transfection reverses the functionalgCSC-mediated immune inhibition of T-cells, anti-CD3/CD28 naïve CD4+T-cells from healthy donors' PBMCs in the presence of gCSC medium wereactivated, 3-day gCSC-conditioned medium from gCSCs transfected withscramble control, miR-124, and miR-124 plus STAT3. The medium fromscrambled miRNA-transfected gCSCs inhibited T-cell proliferation by63.5±13.8% versus 33.0±10.1% in miR-124-transfected gCSCs (n=4, P=0.023)(FIG. 2B). Moreover, fewer apoptotic T-cells were induced by medium frommiR124-tranfected gCSCs than by medium from scramble-transfected gCSCs(FIG. 2C). Next, it was determined whether miR-124 could diminishforkhead box P3 (FoxP3)+ Treg generation induced from naïve CD4+T-cells, mediated by gCSC-conditioned medium. Indeed, the medium frommiR-124-transfected gCSCs led to decreased FoxP3+ T-cell generationcompared with scrambled miRNA-transfected gCSCs (FIG. 2D). Moreover,these were functional Tregs, as assessed by autologous CD4+ T-cellproliferation in coculture assays (FIG. 2E). Furthermore, all theeffects mediated by miR-124 were reversed by cotransfection ofwild-type, constitutively active STAT3 lacking a miR-124 sensitive3′-UTR fragment (FIG. 2B, C, D, E). In contrast, miR-21 enhancedgCSC-mediated immune suppression as assessed by suppression of T-cellproliferation (FIG. 10).

Because miR-124 can modify the immune-suppressive function of gCSCs, itwas determined if it could exert a direct effect on the immune effectorfunction in immunosuppressed glioblastoma patients. PBMCs were obtainedfrom patients newly diagnosed with glioblastoma during tumor resection.The baseline miR-124 expression in glioblastoma patient's T-cells (n=4)and normal donors (n=4) is undetectable when determined by RT-PCR. TheT-cells were stimulated and simultaneously transfected with thescrambled control oligonucleotides or with miR-124. Levels of IL-2,tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were significantlyincreased in miR-124-transfected CD4+ T-cells and CD8+ T-cells (FIG. 3).In parallel, it was also observed that miR-124 overexpression in healthydonor peripheral blood T-cells enhances production of effectorcytokines, such as IFN-γ, TNF-α, and IL-12, from CD4+ and CD8+ T-cells.

4. miR-124 Inhibits In Vivo Glioma Growth

Given miR-124's role in modulating the STAT3 pathway and immuneresponses, whether miR-124 could exert a therapeutic effect in vivo wasdetermined next. To assess the in vivo antitumor efficacy of miR-124,GL261 murine glioma cells were implanted into immune competent C57BL/6mice and treated them with miR-124 or scramble control (n=10 per group).After the subcutaneous GL261 tumors had grown to a palpable size,miR-124 duplex or scramble control was administered. Subcutaneous tumorgrowth progressed in all the C57BL/6J mice treated with the scramblecontrol. In contrast, in the miR-124-treated group, the tumor volume wasmarkedly suppressed (P=0.01) (FIG. 4A). Gliomas started to shrink assoon as miR-124 was administered; moreover, the tumors continued toregress even after miR-124 treatment was discontinued. In contrast,tumors kept growing aggressively in scramble microRNA-treated anduntreated tumor-bearing mice groups. An immunohistochemical analysisrevealed that p-STAT3 glioma expression levels were markedly inhibitedin the miR-124-treated cohort (P=0.0039) (FIG. 4B).

To determine whether enhanced immunological tumor cytotoxicity wascorrelated with miR-124's efficacy in vivo, the immune cytotoxicresponses directed toward GL261 glioma cells were evaluated. Splenocytesfrom tumor-bearing mice treated with miR-124 duplex or scramble miRNAwere isolated and cocultured with CFSE-labeled GL261 target cells for 48hours. The immune cells from the tumor-bearing mice treated withmiRNA-124 increased the cytotoxic clearance of the GL261 target cellsrelative to that in scramble-treated mice (P<0.05) (FIG. 4C and FIG.11). Ex vivo GL261 tumor tissues from miR-124- or scramblemicroRNA-treated tumor-bearing mice were analyzed and found that thepercentage of FoxP3+ Tregs in the tumor microenvironment was reduced to19.0±8.8% in the miR-124-treated group (n=3) compared with 64.7±5.4% inthe scramble-treated group (n=3) (P=0.0015) (one representative FACSplot shown as FIG. 4D). No significant decrease in the number of FoxP3+Tregs in the spleen or lymph nodes of miR-124-treated tumor-bearing micewas observed relative to control-treated mice, indicating that miR-124'sTreg modulatory effects were confined to the tumor. To determine whethermiR-124 mediates an enhanced immune activation of effector T-cells inthe tumor microenvironment, the production of effector cytokines such asIFN-γ and TNF-α in tumor-infiltrating T-cells was determined. Consistentwith the enhanced antitumor activity in the miR-124-treated group, amarked increase in effector cells (i.e., producing IFN-γ or TNF-α) wasfound in the glioma microenvironment, including CD4+ T-cells (FIG. 4E;IFN-γ: from 7.7±2.0% to 21.6±3.3%, P=0.0032; TNF-α: from 6.4±1.7% to29.1±7.4%, P=0.0066) and CD8+ T-cells (FIG. 4F; IFN-γ: from 10.9±3.3% to26.0±4.0%, P=0.007; TNF-α: from 6.4±1.7% to 16.4±1.7%, P=0.0019).

5. The Therapeutic Effect of miR-124 is Immune Mediated

Although, the miR-124 had a therapeutic effect when injected directlyinto the tumor, this is unlikely to be a viable therapeutic approach forpatients. Therefore, intravenous miR-124 administration was tested inestablished murine glioma models. Confirming the results of the directdelivery approach, intravenous administration of miR-124 led to markedinhibition of glioma growth in vivo (FIG. 5A). To determine whether thistherapeutic effect was secondarily mediated by the immune system, GL261murine glioma cells were implanted in immune-incompetent (nude) mice andtreated them with miR-124 or scramble control. Intratumoral treatmentwas initiated when the tumors grew to a palpable size. In theimmune-incompetent animal background, miR-124 failed to exert atherapeutic effect, indicating that miR-124 mediates in vivo activityvia the immune system (FIG. 5B). To determine whether treatment withmiR-124 was effective against established intracerebral tumors, miR-124was administered to C57BL/6J mice with intracerebral tumors from GL261cells, starting after tumor cell implantation. The median survivalduration for the scramble control group was 21.5 days. For mice treatedwith miR-124, the median survival duration was 32 days (P=0.02) (FIG.5C). When the experiment was repeated in an immune-incompetent modelsystem, therapeutic efficacy was once again lost (FIG. 5D).

6. The Immune Therapeutic Efficacy of miR-124 Depends on T-Cells

To further investigate which T-cell compartment mediates miR-124's invivo antitumor activity, CD4+ or CD8+ T-cells in GL261 tumor-bearingmice were depleted with neutralizing antibodies while treating thosemice with miR-124 or scramble RNA oligonucleotides. The depletion ofboth CD4+ T-cells and CD8+ T-cells completely abrogated the anti-gliomaefficacy of miR-124 (FIG. 6A), indicating that CD4+ and CD8+ T-cells arecritical immune cell components mediating miR-124 therapeutic efficacyin vivo. In order to determine whether CD3+ T-cells are directlytargeted during intravenous administration of miR-124, CD3+ T-cells wereisolated from the peripheral blood, spleens and GL261 tumors andmeasured the expression of miR-124 by quantitative RT-PCR. There isminimal baseline expression of miR-124 in the T-cells from non-tumorbearing and GL261-bearing mice (FIG. 12). After in vivo miR-124treatment, there is an increase in the miR-124 expression levels in boththe peripheral blood T-cells and within the glioma-infiltrating T-cells.This coincided with decreased intracellular p-STAT3 expression (FIG.12).

Next, CD3+ T-cells were isolated, transfected with miR-124 or scramblecontrol, and expanded their numbers in vitro for 48 hours beforeadoptively transferring these cells into GL261 tumor-bearing mice. ThismiR-124 transfection inhibited p-STAT3 activity in the adoptivelytransferred T cells (FIG. 6B). Consistent with miR-124-enhanced T-celleffector function (as shown in FIG. 3) and the miR-124 therapeuticeffects relying on T-cells (as shown in FIG. 6A), it was found thatGL261 gliomas regressed upon adoptive transfer of miR-124-transfectedT-cells but not with control scramble-transfected T-cells (FIG. 6C),further demonstrating the pivotal role of the immune system inmiR-124-mediated antitumor effects. To investigate the in vivo cellularmechanisms of adoptively miR-124-transfected T-cell treatment, thepercentage of infiltrating CD4+ T-cells, CD8+ T-cells and FoxP3+ Tregsin the GL261 tumors 6 days after treatment with the miRNA-transfectedCD3+ T-cells was determined. Within the glioma microenvironment, therewas an increase in the CD4+ T-cell infiltration from 2.6±0.9% in thescramble-control transfected CD3+ T-cell treated group to 7.4±1.9% inthe miR-124-transfected CD3+ T-cell treated group (P=0.04, n=3 pergroup), a decrease in FoxP3+ Tregs from 26.9±5.9% to 7.0±0.3% in therespective groups (P=0.014), but no change in the absolute numbers ofCD8+ T-cell infiltration. Similar to the findings in FIGS. 4E and F,there was a marked increase in immune effector cells within the gliomamicroenvironment after treatment with the miR-124-transfected T-cells;specifically, in the CD4+ T-cell compartment (IFN-γ: from 3.7±2.2% inthe scramble-control transfected CD3+ T-cells to 22.5±6.2% in themiR-124-transfected CD3+ T-cells, P=0.023; TNF-α: from 4.1±1.9% to17.2±2.6%, P=0.0076). Although there was no increase in the absolutenumber of CD8+ T-cells, the effector status of the CD8+ T-cells withinthe glioma microenvironment was enhanced (IFN-γ: from 1.4±0.7% to7.3±1.8%, P=0.0018; TNF-α: from 5.2±0.8% to 15±4.4%, P=0.043).

7. miR-124 Modulates T Helper Cell Differentiation

To further investigate whether Th1 and Th17 differentiation areresponsive to modulation with miR-124, CD4+CD45RA+CD45RO− naïve T-cellswere activated with plate-bound anti-CD3 and soluble anti-CD28 underTh1, Th17, and inducible Treg polarization conditions before miR-124transfection. IL-17A+ Th17 cells and FoxP3+ Treg induction was inhibitedwhen miR-124 was overexpressed, whereas miR-124 promoted differentiationof IFN-γ+ Th1 cells (FIG. 13).

8. miR-124 Exerts a Therapeutic Effect in STATS-Expressing GeneticallyEngineered Murine Models

The limitation of evaluating therapeutic strategies in clonotypic modelshas been previously noted (Huse et al., 2009); a genetically engineeredmurine model was created that expresses STAT3 (Doucette et al., 2012).newborn Ntv-a mice were injected with RCAS-STAT3 and RCAS-PDGFB vectorsto reproducibly and consistently obtain high-grade gliomas, with thedefining histologic features of microvascular proliferation, necrosis,and invasion (FIG. 7A) and lacking miR-124 expression (FIG. 7B). Similarto the findings in glioma patients, miR-124 expression in these inducedgliomas was also markedly diminished. To determine whether treatmentwith miR-124 was also efficacious in this model system, Ntv-a mice weretreated with miR-124, starting on day 21 after tumor induction. Nobehavioral or neurological abnormalities of the mice were noted duringtreatment. The median survival duration in the control group was 26days. In mice treated with miR-124, the median survival duration was 39days (P=0.04) (FIG. 7C). Necropsies of glioma-bearing Ntv-a micerevealed that the miR-124-treated cohort had a lower incidence ofhigh-grade gliomas, as determined by the study neuropathologist, on thebasis of the characteristic features of necrosis and neovascularproliferation (FIG. 7D). Furthermore, there was no evidence ofdemyelination, macrophage infiltration, or lymphocytic infiltration inthe non-tumor bearing areas of the CNS that would indicate the inductionof autoimmunity. Systemic administration of miR-124 resulted in lowerp-STAT3 expression in the gliomas than in scrambled miRNA and untreatedcontrols (FIG. 7E).

These experiments demonstrate that miRNA approaches can be exploited forimmune therapeutic purposes against malignancies. A significantconfounding factor in the translational implementation of miR-basedapproaches has been adequate delivery to the target tumor cells. Tocircumvent this, a miRNA that could reverse tumor-mediated immunesuppression—specifically, a key molecular hub, STAT3—resulting inimmunological recognition and clearance of the malignancy was selected.This disclosure also describes a strategy for identifying potentialmiRNA immune therapeutics that may be applicable to other types ofmalignancies by sequentially: 1) screening for down-modulated miRNAsusing tumor microarrays; 2) determining the scope of potential use inhumans by in situ hybridization of tissue microarrays; 3) screening andselecting the miRNA candidates that target immunosuppressive pathwaysand/or mechanisms; and 4) evaluating mechanism and therapeutic effectwithin immune competent model systems. While the STAT3 target was usedas a proof of principal, other immunosuppressive targets such as CTLA-4,PD-1, and transforming growth factor-β could be used. Several othercandidate miRNAs identified in the human glioblastoma miRNA microarrayexpression library likely target several of these as well and are alsobeing evaluated for their potential as therapeutic agents in acomplementary or alternative fashion with miR-124.

The findings support the immune modulatory effects of miR-124. First, invivo therapeutic efficacy was ablated in immune-incompetent murine modelsystems. Second, miR-124 transfection reduced the immune-suppressiveproperties in gCSCs, including inhibiting secretion ofimmune-suppressive cytokines such as galectin-3 (which is downstreamfrom the STAT3 pathway (Wei et al., 2010) and induces T-cell apoptosis,promotes tumor growth, and induces Tregs), MIC-1 (which inhibitsmacrophage production of antitumor TNF-α), and IL-8 (which inducesimmune chemotaxis and is a potent promoter of angiogenesis).Furthermore, inhibition of T-cell proliferative responses and effectorfunctions by the gCSCs was reversed upon transfection with miR-124. Therestoration of T-cell TNF-α effector functions with miR-124 isconsistent with a previous report that STAT3 negatively regulates TNF-α(Murray, 2006). Third, miR-124 treatment of T-cells fromimmune-suppressed glioblastoma patients induced potent effectorresponses, including IL-2 and IFN-γ production. Fourth, the immuneresponses in the glioma microenvironment in miR-124-treated murinemodels demonstrated an enhancement of proinflammatory effector CD4 andCD8 T-cells, with diminished Treg intratumoral trafficking. Finally, exvivo glioma cytotoxicity assays from miR-124-treated mice demonstratedenhanced glioma killing. Cumulatively, these data are consistent withthose of previous studies that demonstrated that modulating the STAT3pathway in the immune cell population is sufficient to mediateefficacious antitumor immune responses (Kortylewski et al., 2005).

STAT3 signaling has been shown to be a key regulator ofmicroglia/macrophage-mediated immune-suppression (Wu et al., 2010).MiR-124 is low or undetectable in these cells; thus miR-124administration may abolish or reverse their immune suppression bydown-regulating STAT3 activity. Although this study focused on adaptiveanti-tumor immune responses, it cannot exclude that part of thetherapeutic effect was mediated via innate immunity. Other investigatorshave shown that the peripheral administration of miR-124 in anexperimental murine autoimmune encephalomyelitis model causeddeactivation of macrophages, reduced activation of myelin-specific Tcells and markedly suppressed the disease (Ponomarev et al., 2011). Thisdiscrepancy can be explained by the contextual target—i.e. miR-124targets overactive C/EBP-α-PU.1 signaling in the context of inducedautoimmunity versus STAT3 signaling in the glioma microenvironment withthe resulting contrasting immune functional differences.

When data from The Cancer Genome Atlas were used to compare miR-124expression and survival in patients with glioblastoma, no differences inpatient outcome were identified; however, the miR-124 expression levelswere negligible in these patients, and the marginal differences areprobably attributable to the submitted specimens containing interveningmiR-124-expressing infiltrating neurons. Given miR-124's role inneuronal development, it was not unexpected to find it expressed in thenormal CNS as assessed by in situ hybridization. miR-124 expression waslost across all grades and types of gliomas, suggesting not only thatthis loss is an early event in glioma initiation and development butalso that miR-124 therapeutic approaches will be useful in a variety ofgliomas.

On the basis of multiple predictive binding algorithms, luciferaseexpression assays, and mutational analyses, miR-124 appears to downregulate the expression of STAT3, including the activated form, p-STAT3.This finding was further supported by the results of in vitro studiesthat demonstrated p-STAT3 inhibition in human gCSCs and immune cells andin vivo in the local glioma microenvironment. These data are alsoconsistent with a recent publication demonstrating that miR-124 binds tothe STAT3 3′-UTR in the rat cardiomyocyte (Cai et al., 2012). However,miR-124 also targets other components of the STAT3 signaling pathwaysuch as Shc1. Although IL-6Rα has been proved to be a target of miR-124in hepatocarcinoma cell lines (Hatziapostolou et al., 2011), this wasnot the case in any of the gCSCs, indicating that miR-124 hasdifferential targets in various cells or tissues. Shc1 is not present innormal brain but is expressed in all grades of gliomas (Magrassi et al.,2005). In glioma patients, the loss of miR-124 may result in theexpression of Shc1, which assembles the EGFR/MAPK1/3 signaling complex,thereby enhancing the activation of this signaling pathway. Because Shc1is upstream of MAPK1/3 in the EGFR/MAPK1/3 signaling pathway, thereduced p-MAPK1/3 level might be due to the down regulation of Shc1 bymiR-124. However, down modulation of Shc1 in most of the gCSCs did notcorrelate with down modulation of p-MAPK1/3. In the one gCSC thatdemonstrated reduced p-MAPK1/3 expression, the IL-6Rα expression wasabsent, indicating a potential greater reliance on the EGFR/MAPK1/3signaling pathway and illustrates that although miR-124 inhibitsp-STAT3, inhibition of other components of the signaling axis arecontextual and hierarchical.

Other than by immune regulation, miR-124 may also reduce gliomagenesisvia multiple mechanisms, including inducing gCSC differentiation,targeting multiple oncogenic signaling pathways (such as NFATc andPIK3CA), and repressing tumor cell proliferation (Lujambio et al.,2007), if sufficient levels of miR-124 are able to enter the CNS. Thedata in the miR-124-treated Ntv-a glioma model demonstrated a decreasedincidence of high-grade glioma, probably secondary to the diminishedp-STAT3 expression in the local tumor microenvironment. This findingconfirms those of previous studies that have linked miR-124 togliomagenesis.

An advantage of intravenous administration of miR-124 is the ease oftranslational implementation as opposed to siRNA approaches that haverequired ex vivo transduction of the cancer cells (Scuto et al., 2011),direct tumor delivery (Bjorge et al., 2011), knock-out in thehematopoietic cell population (Kortylewski et al., 2005), or conjugationto CpG to target the immune population (Kortylewski et al., 2009).Moreover, it is possible that the physiological expression of miR-124 innormal brain tissues confers tolerance to exogenous administration ofthis miRNA, thus minimizing toxicity. Indeed, no evidence of CNStoxicity or induced autoimmunity in treated mice was observed.Alternatively, because miR targets “networks” as opposed to a singulartarget, as is the case with siRNA, other unidentified therapeutictargets may be contributing to the beneficial in vivo effects observedwith the miR-124. Specifically, miR-124 has been previously shown totarget a variety of mRNAs (Lim et al., 2005) and it was found that itcan also target miR-21, which is regulated by STAT3 (Loffler et al.,2007). miR-21 has been shown to be significantly elevated inglioblastomas and can regulate multiple genes associated with preventingglioma cell apoptosis (Chan et al., 2005) and enhancing migration andinvasion (Gabriely et al., 2008). miR-21 inhibition can inhibit thegrowth of glioblastoma cells in vitro (Zhou et al., 2010) and in vivo(Gaur et al., 2011; Corsten et al., 2007). Thus, a component of theobserved in vivo therapeutic effect could be secondary to the modulationof miR-21 by miR-124.

In summary, these findings show that systemic delivery ofimmune-modulatory miRNAs may be used as anticancer therapeutic modality.Immune modulatory miRNAs may be used in combination and delivered in thecontext of nanoparticles, liposomes or exosomes or used to modifycellular vaccine strategies. Because the STAT3 pathway has been shown tomediate resistance to chemotherapeutics by modulating miR-17 (Dai etal., 2011), miR-124 may also have a therapeutic role in the setting oftreatment failure. Screening miRNA expression in tumors used inpersonalized medicine approaches.

Example 2 miR-142-3p Inhibits the M2 Macrophage and Exerts TherapeuticEfficacy Against Murine Glioblastoma

A. Materials and Methods

1. Isolation of Human Glioblastoma-Infiltrating Macrophages andCD14+Monocytes

Tumors were confirmed as glioblastoma (World Health Organization gradeIV) by a board-certified neuropathologist. Tumors were washed in RPMImedium and dissected to remove blood products and surrounding non-tumorbrain tissue. Tumor tissue was broken down into smaller pieces anddigested for 2 hours using a cancer cell isolation kit (Panomics, SantaClara, Calif.). The cells were suspended in RNAlater solution (Ambion,Austin, Tex.) in RNase-free tubes and stored at 4° C. overnight; after24 hours, they were transferred to −20° C. until needed for total RNAextraction. Total RNA from normal brain tissues was obtained fromBiochain (Hayward, Calif.).

After the glioblastomas were placed into single cell suspension, theglioblastoma-infiltrating macrophages were also isolated based onfurther refinement of the glioma-infiltrating macrophage isolationprotocol by performing CD11b+ MACS positive selection after Percollgradient centrifugation to achieve over 95% purity (Hussain et al.,2006). Human PBMCs were prepared from healthy donor blood (n=4) (GulfCoast Blood Center, Houston, Tex.) and from glioblastoma patientsundergoing resection (N=4) at The University of Texas MD Anderson CancerCenter (Houston, Tex.) by centrifugation on a Ficoll-Hypaque densitygradient (Sigma-Aldrich, St. Louis, Mo.). Human CD14+ monocytes werepurified from the PBMCs by anti-CD14 microbeads magnetic cell sorting,according to the manufacturer's instructions (Miltenyi Biotec,Cambridge, Mass.). The purity and viability of the monocytes were >90%.

2. Isolating of RNA and Comparative Analysis

Extraction was performed using the mirVana kit (Ambion, Grand Island,N.Y.). After extraction, the RNA samples were checked for purify andquality via an Agilent Bioanalyzer before being submitted for humanmiRNA array (Sanger miRBase v15, 1,087 human miRNAs) analysis and wholegenome microarray analysis (30,275 human genes) provided by the PhalanxBiotech Group (Belmont, Calif.). The results of the analysis were usedto determine which miRNAs had significant “fold” differences inexpression of: 1) glioblastoma relative to normal brain; 2)glioblastoma-infiltrating macrophages relative to patient matchedmonocytes; 3) glioblastoma-infiltrating macrophages relative to normaldonor monocytes; and 4) glioblastoma monocytes relative to normal donormonocytes, calculated using Microsoft Excel. The miRNAs with the mostsignificant differences in expression levels were then chosen for themiRNA target analysis using multiple bioinformatics prediction tools,including TargetScan 6.2, PicTar, miRanda, and miRDB.

3. Monocyte M1 Versus M2 Differentiation

The M1/M2 in vitro induction protocol was adapted from previous reports(Krausgruber et al., 2011; Sierra-Filardi et al., 2011). Briefly, M1 andM2 macrophages were obtained after 5 days of culture of human CD14+monocytes in RPMI-1640 medium with L-glutamine (Corning Cellgro,Manassas, Va.) supplemented with 10% heat-inactivated FBS(Sigma-Aldrich) and GM-CSF (50 ng/ml for M1) or M-CSF (100 ng/ml for M2;Peprotech, Rocky Hill, N.J.). Phenotypic markers of MHCII, CD11b, CD14,CD45, CD68, CD80, CD86, CD163 and CD206 were confirmed by FACS analysis.

4. Real-Time PCR to Confirm Relative miR-142 Expression Levels

Total RNA extracted from monocytes was used as the template for reversetranscription using the TaqMan real-time PCR kit (Applied Biosystems,Carlsbad, Calif.) in the 7500 real-time PCR system (Applied Biosystems).Primers for reverse transcription were purchased for human miR-142-3pand U18 (Applied Biosystems). U18 was used as an endogenous control andcDNA was used as the template for real-time PCR. Further reactions,substituting water for the cDNA template, were used as additionalcontrols. U18 and miR-142-3p amplifications were run in triplicate.Microsoft Excel was used to calculate the mean levels of each miR andthe U18 internal control. The relative expression levels of miR-142-3pwere compared with those of the internal controls, and bar graphs weregenerated.

5. Luciferase Assay

To determine whether miR-142-3p can bind to TGFβR1 3′-UTR, lipofectamine2000 transfection reagent (Invitrogen, Grand Island, N.Y.) was used toco-transfect HeLa reporter cells with the TGFβR1 3′ UTR-luciferasereporter plasmid (pMirTarget, Origene, Rockville, Md.) and themiR-142-3p expression plasmid (or the scramble control plasmid;GeneCopoeia, Rockville, Md.). The luciferase assay was performed usingthe Dual-Luciferase® reporter assay system (E1910, Promega, Madison,Wis.) and the firefly luciferase activity was normalized to renillaluciferase activity. The interaction between miR-142-3p and its targetwere measured by comparing the results of the co-transfection of theTGFβR13′ UTR-luciferase reporter and the miR-142-3p plasmids with thoseof the 3′ UTR-luciferase reporter plasmid and the scramble controlplasmid.

6. Transfection of miR-142-3p Precursor/Inhibitor into Monocytes

The miR-142-3p precursor, inhibitor, and matching negative controls werepurchased from Ambion. Lipofectamine 2000 transfection reagent(Invitrogen) was used for the transfection of human CD14+ monocytesaccording to the manufacturer's instructions. Briefly, 10⁶ freshlyMACS-sorted human CD14+ monocytes were incubated in a 12-well platecontaining 800 μL of either M1 or M2 differentiation medium withoutantibiotics for 2 hours. The miRNA precursor/inhibitor/matching control(100 pmol) and 2 μL of lipofectamine 2000 reagent in 198 μL Opti-MEM Imedium were mixed gently and then incubated for 20 min at roomtemperature. The final concentration of miRNA was 100 nM, and thetransfection efficiency was confirmed by qRT-PCR.

7. Macrophage Apoptosis Assay

After the transfection of the miR-142-3p precursor and matching negativecontrols with lipofectamine 2000 transfection reagent, CD14+ monocyteswere cultured in either the M1 or M2 differentiation medium. After 24 hand 48 h, the cells were harvested and labeled with Annexin V-PE and7-amino-actinomycin D according to the manufacturer's instructions (BDPharmingen, San Diego, Calif.). The total cell apoptosis was analyzed byflow cytometry within 1 hour. To ascertain the relationship betweenTGFβR1 blockade and apoptosis, untransfected M1 or M2-committedmonocytes were incubated with titrated concentrations of two TGFβR1inhibitors, SB431542 (Chen et al., 2008) and LY-364947 (Hardee et al.,2012) (EMD Millipore; Billerica, Mass.) for 48 hours. SB431542 is aspecific inhibitor of the TGF-β superfamily type I activin receptor-likekinase receptors ALK4, ALK5 and ALK7 (Inman et al., 2002) whereasLY-364947 has been shown to have cross reactivity with VEGF (Vogt etal., 2011). Moreover, M1 or M2-committed monocytes were also transfectedwith two different TGFβR1 siRNAs (set #1 sense: GGCAUCAAAAUGUAAUUCUtt(SEQ ID NO:8), antisense: AGAAUUACAUUUUGAUGCCtt (SEQ ID NO:9), finalconcentration: 100 nM; set #2 sense: CCAUUGAUAUUGCUCCAAAtt (SEQ IDNO:10), antisense: UUUGGAGCAAUAUCAAUGGta (SEQ ID NO:11), finalconcentration: 5 nM) with matched controls (Ambion) by Lipofectamine2000 transfection reagent (Invitrogen). The knockdown efficiency ofTGFBR1 mRNA was confirmed by qRT-PCR.

8. In Vivo Experiments

The miR-142-3p duplex that mimics pre-miR-142-3p (sense:5′-UGUAGUGUUUCCUACUUUAUGGAU-3′ (SEQ ID NO:12), antisense:5′-CCAUAAAGUAGGAAACACUACAAA-3′ (SEQ ID NO:13)) and the scramble controlmiRNA duplex (sense: 5′-AGUACUGCUUACGAUACGGTT-3′ (SEQ ID NO:14),antisense: 5′-CCGUAUCGUAAGCAGUACUTT-3′ (SEQ ID NO:15)) were synthesized(Avetra Bioscience, San Carlos, Calif.). The sequence of murinemiR-142-3p is identical to human miR-142-3p on the basis of NCBI blastdata. The treatment cohorts consisted of 20 μg of the miR-142-3p orscramble control in 48 μL of phosphate-buffered saline (PBS) mixed withthe vehicle (40 PBS containing 10 μL lipofectamine 2000) or the vehiclecontrol (90 μL PBS+10 lifofectamine 2000). Mice were maintained in theM.D. Anderson Isolation Facility in accordance with Laboratory AnimalResources Commission standards and handled according to the approvedprotocol 08-06-11831.

9. Syngeneic Subcutaneous Model

The murine glioma GL261 cell line was obtained from the National CancerInstitute-Frederick Cancer Research Tumor Repository. These cells werecultured in a humidified atmosphere of 5% CO₂ and 95% air at 37° C. inDulbecco's Modified Eagle's medium (Life Technologies; Grand Island,N.Y.) supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin/L-glutamine (Mediatech, Manassas, Va.). TheGL261 glioma cell cultures were passaged every 3 days to ensurelogarithmic growth. To induce subcutaneous tumors, logarithmicallygrowing GL261 cells were injected into the right hind flanks of6-week-old C57BL/6J female mice or nude mice at a dose of 1×10⁶ cellssuspended in 100 μL of matrigel basement membrane matrix (BDBiosciences, San Jose, Calif.). When palpable tumors formed that wereapproximately 0.5 cm in diameter, the mice (n=5/group) were treated byeither local tumor injection or intravenous administration. Tumors weremeasured twice per week. Mice that showed signs of morbidity, high tumorburden, or skin necrosis were immediately euthanized according to M.D.Anderson guidelines. Tumor volume was calculated with slide calipersusing the following formula: V=(L×W×H)/2, where V is volume (mm³), L isthe long diameter, W is the short diameter, and H is the height.

10. Syngeneic Intracranial Clonotypic Glioma Model

To induce intracerebral tumors in C57BL/6J mice, GL261 cells werecollected in logarithmic growth phase, washed twice with PBS, mixed withan equal volume of 10% methyl cellulose in improved modified Eagle'smedium (zinc option; Life Technologies, Inc., Gaithersburg, Md.), andloaded into a 250-μ1 syringe (Hamilton, Reno, Nev.) with an attached25-gauge needle. The mice were anesthetized with ketamine (100 mg/kg)and xylazine (10 mg/kg), and the needle was positioned 2 mm to the rightof bregma and 4 mm below the surface of the skull at the coronal sutureusing a stereotactic frame (Kopf Instruments, Tujunga, Calif.), aspreviously described (Heimberger et al., 2003). The intracerebraltumorigenic dose for GL261 cells was 5×10⁴ in a total volume of 5 μl.Mice were then randomly assigned to scramble control (n=6) or themiR-142-3p treatment group (n=7). Animals were observed and weightedthree times per week, and when they showed signs of neurological deficit(lethargy, failure to ambulate, lack of feeding, or loss of >20% bodyweight), they were compassionately euthanized. These symptoms typicallyoccurred within 48 hours of death. Their brains were removed and placedin 4% paraformaldehyde and embedded in paraffin. The intracerebraltumorigenic dose for GL261 cells was 5×104 in a total volume of 5 μl.Mice were randomly assigned to scramble control or the miR-142-3ptreatment group. Animals were observed and weighed three times per week,and when they showed signs of neurological symptoms, they werecompassionately euthanized. Their brains were removed, placed in 4%paraformaldehyde and embedded in paraffin.

11. Genetically Engineered Murine Models

Vector constructs. RCAS-PDGFB generation has been previously described(Dai et al., 2001). DF-1 immortalized chicken fibroblasts were grown inDulbecco's modified Eagle's medium with 10% fetal bovine serum in ahumidified atmosphere of 95% air/5% CO2 at 37° C. Live virus wasproduced by transfecting plasmid versions of RCAS vectors into DF-1cells using FuGene6. These cells were replicated in culture.

In vivo Somatic Cell Transfer in Transgenic Mice. The transgenic Ntv-amice are mixtures of different strains, including C57BL/6, BALB/c,FVB/N, and CD1. To transfer genes via RCAS vectors, DF-1 producer cellstransfected with a particular RCAS vector (1×10⁵ DF-1 cells in 1-2 μl ofPBS) were injected into the frontal lobes of Ntv-a mice at the coronalsuture of the skull using a Hamilton Gastight syringe. The mice wereinjected on postnatal days 1 or 2, when the number of Nestin+ cellsproducing TVA is the highest. The mice were killed 90 days afterinjection or sooner if they demonstrated morbidity related to tumorburden. Their brains were removed and analyzed for tumor formation.

Animal Randomization. Twenty-one days after introducing theglioma-inducing transgenes RCAS-PDGFB and RCAS-BCL2, littermates wererandomly assigned to the treatment or control group (n=9/group). Micewere treated intravenously on Monday, Wednesday, and Friday for 3 weeks.

12. Immunohistochemistry

Formalin-fixed, paraffin-embedded 4 μm sections of the glioma were firstdeparaffinized in xylene and rehydrated in ethanol. Endogenousperoxidase was blocked with 0.3% hydrogen peroxide/methanol for 10 minat room temperature. Then, the ThermoScientific PTModule (Thermo FisherScientific, Fremont, Calif.) with citrate buffer (pH 6.0) was used forantigen retrieval. Immunohistochemical staining was performed using theLab Vision Immunohistochemical Autostainer 360 (Thermo FisherScientific, Fremont, Calif.). The staining was visualized using anavidin-biotin complex technique with diaminobenzidine (Invitrogen,Carlsbad, Calif.) as the chromogenic substrate and hematoxylin as thecounterstain. To detect expression of the macrophage-restricted cellsurface glycoprotein F4/80, a purified anti-mouse F4/80 (1:50;Biolegend, San Diego, Calif.) antibody was used. Two independentobservers (L-YK, X-Y L) quantitatively evaluated F4/80 expression byanalyzing the tumors using high-power fields (max: ×40 objective and ×10eyepiece) of each specimen. The observers were blinded to the treatmentcohorts and the analysis was secondarily reviewed again by theneuropathologist (GNF). The correlation between miR-142-3p treated micesurvival and corresponding F4/80 positive cell percentages was fittedwith linear regression using GraphPad Prism (La Jolla, Calif.).

13. Isolation of Human Glioblastoma-Infiltrating Macrophages andCD14+Monocytes

Informed consent was obtained from each subject. Tumors were confirmedas glioblastoma (World Health Organization grade IV) by aboard-certified neuropathologist. Tumor tissue was washed in RPMImedium, broken down into smaller pieces and digested for 2 hours using acancer cell isolation kit (Panomics, Santa Clara, Calif.). The techniquefor isolating central nervous system macrophages based on CD11bexpression and Percoll gradient centrifugation in mice (Stupp et al.,2005; miR-142 manu) was adapted for isolating human glioblastomainfiltrating macrophages by our group (Hussain et al., 2006) andconsisted of CD11b+ MACS positive selection after Percoll gradientcentrifugation to achieve over 95% purity. These purifiedglioblastoma-infiltrating macrophages express CD14 (Hussain et al.,2006). Matched peripheral monocytes and monocytes from the healthydonors were obtained with anti-CD14 [a general marker of monocytes andmonocyte-derived macrophages (Wu et al., 2010)] microbead magnetic cellsorting, according to the manufacturer's instructions (Miltenyi Biotec,Cambridge, Mass.). CD14+ monocytes have previously been shown to beinduced to the M2 phenotype, similar to glioblastoma-infiltratingmacrophages, by supernatants from glioblastoma cancer stem cells(Gabriely et al., 2008).

14. Isolating of RNA and Comparative Analysis

After extraction using the mirVana kit (Ambion, Grand Island, N.Y.), theRNA samples from infiltrating macrophages, peripheral monocytes andtotal glioblastoma specimens were checked for purify and quality via anAgilent Bioanalyzer before being submitted for human miRNA array (SangermiRBase v15, 1,087 human miRNAs) analysis and whole genome microarrayanalysis (30,275 human genes) provided by the Phalanx Biotech Group(Belmont, Calif.). This microarray data has been submitted to the NCBIGEO database (assession number is GSE51332). Total RNA from normal braintissues was obtained from Biochain (Hayward, Calif.). The results of theanalysis were used to determine which miRNAs had significant “fold”differences in expression of: 1) glioblastoma relative to normal brain;2) glioblastoma-infiltrating macrophages relative to patient matchedmonocytes; 3) glioblastoma-infiltrating macrophages relative to normaldonor monocytes; and 4) glioblastoma monocytes relative to normal donormonocytes, calculated using Microsoft Excel. The miRNAs with the mostsignificant differences in expression levels were then chosen for themiRNA target analysis using multiple bioinformatics prediction tools,including TargetScan 6.2, PicTar, miRanda, and miRDB.

15. Human gCSCs

gCSCs were cultured in Dulbecco's modified Eagle's medium F-12containing 20 ng/ml of epidermal growth factor, basic fibroblast growthfactor (both from Sigma, St. Louis, Mo.), and B27 (1:50; Invitrogen,Carlsbad, Calif.) as a neural stem cell-permissive medium (neurospheremedium) and passaged every 5-7 days. The characteristics of these cells,including the cytogenetics, limiting dilution assays, tumorigenicity,CD133 expression, and immune-suppressive properties have been previouslypublished (Gabriely et al., 2008; Iliopoulos et al., 2010).

16. Monocyte to M1 Versus M2 Macrophage Differentiation

The M1/M2 in vitro induction protocol was adapted from previous reports(Wang et al., 2012; Hui et al., 2010). Briefly, M1 and M2 macrophageswere obtained after 5 days of culture of human CD14+ monocytes inRPMI-1640 medium with L-glutamine (Corning Cellgro, Manassas, Va.)supplemented with 10% heat-inactivated FBS (Sigma-Aldrich) and GM-CSF(50 ng/ml for M1) or M-CSF (100 ng/ml for M2; Peprotech, Rocky Hill,N.J.). Phenotypic markers of immune status (MHC, CD45, CD80 and CD86)monocyte-macrophage lineage (CD14), macrophage (CD11b, CD68), and M2differentiation (CD163) were confirmed by FACS analysis.

17. Phagocytotic Assay

The phagocytic activity of the M1/M2 macrophages was determined bymeasuring the uptake of fluorescent pHrodo Red E. coli bioparticles(Life Technologies, Carlsbad, Calif.). Briefly, the fluorescentbioparticles were resuspended in uptake buffer at a concentration of 1mg/ml and sonicated to homogeneously disperse the particles. Then themedium of pre-seeded M1/M2 macrophages in 96-well plates (105 cells perwell) was removed and replaced with the bioparticle suspension. After 2hours of incubation at 37° C., the fluorescent intensity was measured bya fluorescence plate reader (BMG LabTech GmbH, Ortenberg, Germany) at580 nm. The net phagocytosis is calculated by subtracting theappropriate negative/blank control fluorescent intensity from theexperimental wells.

18. Western Blot

M1 and M2 macrophages were harvested on day 5 after transfection withpre-miR-142-3p and then lysed in RIPA cell lysis buffer supplementedwith a protease inhibitor cocktail (P8340, Sigma-Aldrich) and aphosphatase inhibitor (P5726, Sigma-Aldrich). Protein concentration wasdetermined with the BCA protein assay kit (Pierce). Twenty micrograms ofprotein of each sample were separated by 4-15% sodium dodecyl sulfate(SDS)-polyacrylamide gels (Bio-Rad) and electro-transferred tonitrocellulose membranes, and subjected to immunoblot analysis withantibodies to ITGB8 (1:500, Sigma-Aldrich), a-Tubulin (1:2000,Sigma-Aldrich), ITGAV (1:500, Santa Cruz, Dallas, Tex.), TGFβ2 (1:1000,Santa Cruz), SMAD4 (1:1000, Santa Cruz), TGFβR1 (1:1000, Santa Cruz),NF-κB (1:1000, Cell Signaling, Danvers, Mass.), β-Actin (1:1000, CellSignaling), p-SMAD2/3 (1:1000, Cell Signaling), p-Akt (1:1000, CellSignaling), p-TAK1 (1:1000, Cell Signaling) and RhoA (1:1000 CellSignaling). Autoradiography of the membranes was performed usingAmersham ECL Western-blotting detection reagents (Amersham Biosciences,Piscataway, N.J.).

19. ELISA

Supernatant medium conditioned by macrophages transfected withmiR-142-3p or scramble control was measured for TGF-β2 using ELISAs(DuoSet ELISA kits, R&D Systems). The supernatants were collected after48 hours and 5 days in culture and stored at −20° C. Beforequalification, the supernatants were pre-treated with 1N HCl to activatethe immunoreactive form, and then added in triplicate to appropriatecapture antibody-coated plates. After the plates were washed,horseradish peroxidase-conjugated detection antibody was added. Thesubstrate used for color development was tetramethylbenzidine. Theoptical density was measured at 450 nm with a microplate reader (SpectraMax 190; Molecular Devices, Sunnyvale, Calif.) and cytokineconcentrations were quantified with SoftMax Pro software (MolecularDevices).

20. Luciferase Assay

To determine whether miR-142-3p can bind to TGFBR1 3′-UTR, thelipofectamine 2000 transfection reagent (Invitrogen, Grand Island, N.Y.)was used to co-transfect HeLa cells with the TGFBR1 3′ UTR-luciferasereporter plasmid (BlueHeron, Bothell, Wash.), pRL-TK renilla luciferaseplasmid (Promega, Madison, Wis.), and the miR-142-3p precursor. Theluciferase assay was performed using the Dual-Luciferase® reporter assaysystem (Promega, Madison, Wis.) and the firefly luciferase activity wasnormalized to renilla luciferase activity. The interaction betweenmiR-142-3p and its target were measured by comparing the fireflyluciferase activity in the presence or absence of miR-142-3p which wasnormalized by the renilla luciferase activity.

21. Macrophage Apoptosis Assay

After the transfection, CD14+ monocytes were cultured in either the M1or M2 differentiation medium. After 48 hours, the cells were harvestedand labeled with Annexin V-PE and 7-amino-actinomycin D according to themanufacturer's instructions (BD Pharmingen, San Diego, Calif.). Thetotal cell apoptosis was analyzed by flow cytometry within 1 hour. Toascertain the relationship between TGFBR1 blockade and apoptosis,untransfected M1 or M2-committed macrophages were incubated withtitrated concentrations of two TGFBR1 inhibitors, SB431542 (Chan et al.,2011; Wu et al., 2012) and LY-364947 (Inoue et al., 2012; Namlos et al.,2012) (EMD Millipore, Billerica, Mass.) for 48 hours. M1 or M2-committedmacrophages were transfected with two different TGFBR1 siRNAs (set #1sense: GGCAUCAAAAUGUAAUUCUtt (SEQ ID NO:16), antisense:AGAAUUACAUUUUGAUGCCtt (SEQ ID NO:17), final concentration: 100 nM; set#2 sense: CCAUUGAUAUUGCUCCAAAtt (SEQ ID NO:18), antisense:UUUGGAGCAAUAUCAAUGGta (SEQ ID NO:19), final concentration: 5 nM) withmatched controls (Ambion). The knockdown efficiency of TGFBR1 mRNA wasconfirmed by qRT-PCR.

22. Pharmacokinetic Study

Non-tumor bearing C57BL/6J mice (n=3 per time point) were administeredmiR+lipofectamine 2000 i.v. once and subsequently terminated at 0, 15minutes, 1, 4, 8 and 24 hours. The liver, peripheral blood mononuclearcells and serum were subsequently analyzed for miR expression byquantitative PCR. A noncompartmental analysis was performed usingindustry standard software (WinNonLin 6.3, Pharsight) to estimate thepharmacokinetic parameters for each individual animal. The followingparameters were estimated for each animal: apparent eliminationhalf-life (T1/2, calculated as ln(2)/lambdaz, lambdaz being the firstorder rate constant associated with the terminal portion of thetime-concentration curve as estimated by linear regression of time vs.log concentration), area under the time-concentration curve from timezero to the last observed concentration (AUC0-obs, calculated by thelinear trapezoidal rule), and area under the time-concentration curvefrom time zero extrapolated to infinity (AUC0-inf, calculated by addingthe last observed concentration divided by lambda z to the AUC0-obs).Mean parameters were then calculated from individual animal estimates.

23. Ex Vivo Immune Functional Analysis

GL261 tumor-bearing mice were treated with miR-142-3p or scramblecontrol as previously described. After 3 days, the blood, lymph nodesand spleens were harvested and single cell suspensions were obtained.For M1 and M2 marker analysis, the cells were surface stained withCD11b, permeabilized with Cytofix/Cytoperm (BD Pharmingen), and thenstained with FITC conjugated anti-IL-6, anti-IFN-γ or anti-TNF-α (BDPharmingen). Control isotype antibodies were used to establish gatingand were analyzed using FlowJo software (TreeStar). For analysis of CD8+T-cell effector function, GL261 tumor-bearing mice were treated for twoweeks, and CD8+ T cells were purified from the splenocytes by negativeselection using CD8+ T cell enrichment microbeads (BD Biosciences). Forintracellular cytokine staining, the T-cells were stimulated for 6 hoursin the presence of 50 ng/ml phorbol myristate acetate (PMA), 500 ng/mlionomycin (Sigma-Aldrich), and 2 μM monensin (Golgi Stop, BD Sciences),permeabilized, and then stained using either PE-conjugated IFN-γantibody, IL-2, or TNF-α (BD Pharmingen). For ex vivo cytotoxicityassay, the ratios of CD8 T-cells to 2 μM CSFE-labeled GL261 target cellswere 1:1, 5:1, 10:1 and 20:1. Viability of CFSE-labeled GL261 cells wasassessed using propidium iodide staining and a FACS Calibur flowcytometer (BD Biosciences).

24. Mature Dendritic Cell Analysis

Single cell suspensions were prepared from the spleens of miR-142-3p orscramble control treated GL261 tumor-bearing mice. Splenocytes werestained with the PE-labeled anti-mouse dendritic cell marker (33D1) andthe APC-labeled anti-MHCII (I-A/I-E) (eBioscience) and acquired on aFACS Calibur (Becton Dickinson) to determine the percentage of33D1+MHCII+mature dendritic cells.

25. Statistical Analysis

Linear mixed models were fit to assess tumor growth after adjusting fortreatment effect and taking into account the associations among repeatedmeasures within each subject. Kaplan-Meier curves were used to estimateoverall survival (OS) distributions. Log rank tests were used to compareOS between groups. All computations were carried out in SAS version 9.3(SAS Institute, Cary, N.C.) and TIBCO Spotfire S+ version 8.2(Somerville, Mass.).

B. Results

1. miR-142-3p Expression is Down-Modulated in Monocyte-DerivedGlioblastoma-Associated Macrophages

Although monocyte-derived glioblastoma-infiltrating macrophages havebeen well investigated for their immune suppressive properties, therelationship of this most abundant immune cell population within theglioblastoma microenvironment to miRNA dysregulation has not beenevaluated to date. Utilizing the Human miRNA OneArray Microarray v2 toassess miRNA expression profiling, the miRNA expression profile inglioblastoma-infiltrating macrophages was matched to that of monocytesfrom the peripheral blood, and significantly modulated miRNAs wereidentified. With a mean 4.9-fold decrease relative to the level inmatched peripheral monocytes, miR-142-3p emerged as a leading candidate(FIG. 14A and Table 5). On a microarray study of total miRNA, downregulation of miR-142-3p down-regulation wasn't observed in glioblastomatissue relative to normal brain tissue (Table 6). This may be explainedby the subsequent quantitative RT-PCR analysis, which revealed thatdespite the fact that miR-142-3p expression is detected in totalglioblastoma tumor tissues, glioblastoma cell lines (such as U-87 MG andU251) or glioma cancer stem cells (gCSCs) seldom express this miRNA. Onthe contrary, non-tumor cells such as monocytes are the main source ofmiR-142-3p, but the miR-142-3p is down regulated within theglioblastoma-infiltrating macrophages relative to peripheral bloodmonocytes (FIG. 14B). Of note, there was no significant difference ofmiR-142-3p expression in peripheral monocytes from patients withglioblastoma compared to healthy donors.

TABLE 5 miRNA expression in glioblastoma-infiltrating macrophagesrelative to that in peripheral blood monocytes Relative down Relative upmiRNA regulation miRNA regulation hsa-miR-142-3p 5.0 hsa-miR-4792 8.6hsa-miR-92a 4.9 hsa-miR-574-5p 6.4 hsa-miR-15a 4.4 hsa-miR-34a 6.4hsa-miR-92b 3.8 hsa-miR-4290 6.0 hsa-miR-378 3.8 hsa-miR-3149 5.7hsa-miR-16 3.7 hsa-miR-4455 5.6 hsa-miR-636 3.7 hsa-miR-32* 5.6hsa-miR-20b 3.6 hsa-miR-1273f 4.7 hsa-miR-378i 3.6 hsa-miR-4634 4.6hsa-miR-106a 3.6 hsa-miR-3653 4.1 hsa-miR-142-5p 3.6 hsa-miR-634 4.1hsa-miR-20a 3.5 hsa-miR-2116* 3.9 hsa-miR-19b 3.4 hsa-miR-4323 3.9hsa-miR-18a 3.3 hsa-miR-1273e 3.8 hsa-miR-18b 3.3 hsa-miR-532-3p 3.7hsa-miR-93 3.3 hsa-miR-4508 3.6 hsa-miR-17 3.3 hsa-miR-92b* 3.4hsa-miR-19a 3.3 hsa-miR-4713-5p 3.3 hsa-miR-425 3.3 hsa-miR-30c-1* 3.3hsa-miR-223* 3.2 “hsa-miR-4492/4508” 3.3

TABLE 6 Expression of miRNAs in glioblastoma relative to normal brainRelative down Relative up miRNA regulation miRNA regulation hsa-miR-12424.6 hsa-miR-1273 3.7 hsa-miR-3172 13.8 hsa-miR-559 3.6 hsa-miR-138 13.4hsa-miR-4286 3.2 hsa-miR-3196 8.5 hsa-miR-3152 3.1 let-7b 7.3hsa-miR-766 3 let-7e 6.9 hsa-miR-542-3p 2.7 hsa-miR-1826 5.9hsa-miR-1302 2.7 hsa-miR-1228* 5.8 hsa-miR-2355 2.7 hsa-miR-4284 5.6hsa-miR-1285 2.6 let-7d 5.6 hsa-miR-548c-5p 2.5 hsa-miR-3162 5.4hsa-miR-1281 2.3 hsa-miR-874 5.2 hsa-miR-1248 2.2 let-7c 5.2hsa-miR-1272 2.1 hsa-miR-103 5 hsa-miR-488* 2.1 hsa-miR-128 4.9hsa-miR-3192 2.1 let-7a 4.7 hsa-miR-548d-5p 2.1 hsa-miR-26a 4.5hsa-miR-3197 2.1 hsa-miR-762 4.5 hsa-miR-4323 2.1 hsa-miR-7 4.2hsa-miR-3146 2 hsa-miR-142-3p 1.2

2. miR-142-3p Expression in M1 Vs. M2 Macrophages

Glioblastomas actively recruit macrophages to the tumor site and inducethem to adopt a tumor-supportive M2 phenotype capable of mediatingimmunosuppression and promoting invasion (Wu et al., 2010). Based on theobservation of miR-142-3p down-regulation in glioblastoma-infiltratingmacrophages, the miR-142-3p expression in the proinflammatory M1 subsetand immunosuppressive M2 subsets were clarified. Peripheral human CD14+monocytes from healthy donors were isolated and incubated them incomplete RPMI medium supplemented with recombinant human GM-CSF or M-CSFto induce M1 and M2 macrophages, respectively. After 5 days of in vitroinduction, the two subsets were obtained and verified based on theirmorphological, phenotypic, and phagocytic characteristics. Specifically,the M1 macrophages appeared more round, whereas the M2 macrophagesassumed a more elongated phenotype as previously described (FIG.15A)(Hashimoto et al., 1999). Moreover, the M2 macrophages expressedlower levels of CD11b, CD86, and MHC II than the M1 macrophages but hadhigher levels of CD163 (FIG. 15B) and exhibited more dynamicphagocytosis (P<0.001, n=6, FIG. 15C), consistent with prior reports(Chihara et al., 2012; Heusinkveld et al., 2011). The down-regulation ofmiR-142-3p during monocyte to macrophage differentiation was confirmedby qRT-PCR assay; however, this alteration was more profound in theimmune suppressive M2 macrophages (p<0.05, FIG. 15D).

3. miR-142-3p Interacts with the TGF-β Pathway

Multiple bioinformatics tools (TargetScan 6.2, PicTar, miRanda andmiRDB) were used to identify potential target genes of miR-142-3p. Acluster of TGF-β pathway genes with conserved target sites in their3′-UTRs were identified, including TGFB2/3, TGFBR1, and TAB2. The TGF-βpathway is well known for its potent role in immunosuppression andtumorigenesis including polarization of tumor-promoting M2 macrophages(Flavell et al., 2010). Thus, the effect of miR-142-3p on the expressionof the TGF-β pathway in the monocyte-derived M1 and M2 subsets wasinvestigated. After transfection with miR-142-3p, the human CD14+monocytes were polarized to the M1 and M2 subsets using standard ex vivoculture techniques described above. The miR-142-3p overexpression wasconfirmed compared to blank and scramble control transfected cells usingqRT-PCR (FIG. 20A). In the miR-142-3p-overexpressing cells, the mRNAlevel of TGFβR1 mRNA remained unchanged (FIG. 20B); whereas the TGFβR1protein levels were repressed in the M2 macrophages as shown by Westernblot (FIG. 16A) indicating that miR-142-3p mediates its effect on theTGFβR1 pathway by post-transcriptional regulation rather than bytargeting mRNA degradation. TGF-β2 cytokine secretion was not inhibitedby miR-142-3p in either the M1 or M2 macrophages (FIG. 20A-B).Furthermore, anti-miR-142-3p treatment resulted in slightly increasedTGFβR1 protein levels in the M2 macrophages (FIG. 16A) signifying thatmiR-142-3p TGF-β pathway regulation is contextual and cell specific.Other predicted targets of miR-142-3p such as ITGB8, ITGAV, and TGFβ2were not found to be inhibited in either the M1 or M2 macrophagepopulations (FIG. 16A). Furthermore, we detected the activation of theTGFBR1 downstream protein, SMAD2, after stimulation of M1 and M2 cellswith TGFβ1. As expected, p-SMAD2 was inhibited by miR-142-3p only in M2macrophages but not in the M1 macrophages after stimulation with TGFβ-1(FIG. 16B). TGFBR1 SMAD-independent targets such as p-AKT and RhoA werenot appreciably altered by miR-142-3p. p-TAK1 was not observed to beexpressed in either M1 or M2 populations.

4. TGF-b Stimulus Assay-Smad2/3/4-pSmad2/3/4 Pending

To further prove that miR-142-3p binds to TGFβR1, a luciferaseexpression assay was conducted, including mutating the predictedmiR-142-3pTGFβR13′-UTR binding site (FIG. 16B). In co-transfected HeLacells, TGFβR1 luciferase activity was significantly inhibited bymiR-142-3p, whereas directed mutational alteration of the miR-142-3pTGFβR13′-UTR binding site resulted in partial abolishment of luciferaseactivity (FIG. 16C). However, in co-transfected M2 cells, directedmutational alteration of the miR-142-3p TGFβR13′-UTR binding siteresulted in complete abolishment of luciferase activity (FIG. 16D)emphasizing that TGFβR1 is under the regulation of miR-142-3p in M2macrophages.

5. Overexpression of miR-142-3p Minimally Induces a Phenotypic Shiftfrom M2 to M1 Macrophages

The miR-142-3p overexpression alters the monocyte-derived macrophagephenotype and function was assessed next. CD68, a general macrophagemarker, and CD163, a M2 specific marker (Jensen et al., 2009; Pander etat., 2011; Puig-Kroger et al., 2009), were selected to evaluate thephenotypic shift. After the miR-142-3p or inhibitor transfection, CD68levels of both the M1 and M2 macrophages were unchanged (ctrl: 87.8±5.1%versus miR: 87.8±5.1%, P=0.9987, n=8, FIGS. 21A and 21C), suggestingthat miR-142-3p doesn't influence monocyte to macrophagedifferentiation. Although inconsistently observed, CD163 expression wasdown-regulated upon the overexpression of miR-142-3p, indicating thismiR can modestly modulate the M1/M2 shift and push macrophagedifferentiation to the pro-inflammatory M1 subset (ctrl: 74.6±2.8%versus miR: 63.9±6.0%, P=0.02, n=8, FIGS. 22B and 22C).

6. miR-142-3p Overexpression Induces Selective Apoptosis in M2Macrophages Via TGF-β Blockade

During in vitro culture of the monocytes and differentiated macrophageswith miR-142-3p, a significant decrease in the relative cell number ofthe M2 macrophages was observed. Specifically, there was more early-(Annexin 7-AAD⁻) and late-apoptosis/cellular death (Annexin 7-AAD⁺)induced in M2-relative to M1 committed-monocytes at 48 hours aftermiR-142-3p transfection. The fold of change induced by miR-142-3poverexpression in M2 macrophages was 2.58±0.56, compared to 1.24±0.13 inthe M1 macrophages (P=0.02, FIG. 17A). Because TGF-β has been shown tohave a role in autocrine/paracrine growth of cells (Muraoka et al.,2002), TGFβR1 blockade in the M2 cells was investigated next, which mayhave autocrine dependency on this pathway, would induce selectiveapoptosis. Therefore, both M1- and M2-committed monocytes were treatedwith the TGFβRI inhibitors SB431542 and LY364947 to mimic the blockadepotentially triggered by miR-142-3p transfection. Again, more apoptosiswas observed within M2-committed monocytes treated with eitherantagonist than in the M1-committed monocytes (P=0.04 and 0.001,respectively; FIG. 4B). Moreover, knockdown of the TGFβR1 mRNA levels inM1/M2-committed macrophages with specific TGFβR1 siRNAs (FIG. 23A),induced preferential apoptosis in the M2 subset (FIG. 23B). All thesedata indicate that miR-142-3p overexpression induces selective apoptosisin M2 macrophages by blockade of the TGF-β receptor signaling pathway.Of note, this appears to be cell type specific since transfection ofgCSCs with miR-142-3p did not induce significant apoptosis (FIG. 22D).

7. miR-142-3p Inhibits In Vivo Glioma Growth

Considering the role of miR-142-3p in inducing M2 apoptosis bydisruption of TGF-β receptor signaling pathway, miR-142-3p could exertan anti-tumor effect in vivo was investigated next. GL261 murine gliomacells were implanted subcutaneously into immune competent C57BL/6 mice.After the tumor mass had grown to a palpable size, either a scrambledmiRNA sequence or miR-142-3p was administered locally (n=5 per group,FIG. 18A). As shown in FIG. 5B, aggressive tumor growth was observed inthe scramble control group. In contrast, glioma growth was inhibitedduring the 3-week course of miR-142-3p treatment (P=0.03). All the micewere euthanized upon completion of the treatment course, and the tumortissues were resected for H&E staining to confirm tumor formation.Subcutaneous tumors were found in only one out of five mice treated withthe miR-142-3p duplex.

To determine whether miR-142-3p treatment can exert a similar anti-tumoreffect against established intracerebral glioma, C57BL/6J mice harboringintracranial GL261 tumors were treated with miR-142-3p intravenously for3 weeks (n=10 per group, FIG. 18C). The median survival duration for thescramble control group was 23.5 days, which was extended to 31 days formice treated with miR-142-3p (P=0.03) (FIG. 18D). During the treatmentcourse, no behavioral or neurological abnormalities were observed in themice. Necropsies of glioma-bearing mice by the study neuropathologistdemonstrated no evidence of demyelination, macrophage infiltration, orlymphocytic infiltration in the non-tumor-bearing areas of the CNS thatwould indicate the induction of autoimmunity.

The limitation of evaluating therapeutic strategies in clonotypic modelshas been previously noted (Huse et al., 2009). In a geneticallyengineered murine model system of high-grade glioma, it has beenpreviously found that the gliomas have a marked influx of macrophages(Kong et al., 2010). Therefore, newborn Ntv-a mice were injected withRCAS-Bcl-2 and RCAS-PDGFB vectors and subsequently treated them withscramble control or miR-142-3p (FIG. 18E). The median survival durationin the control group was 24 days. In mice treated with miR-142-3p, themedian survival duration was 32 days (P=0.03) (FIG. 18F).

8. miR-142-3p Inhibits In Vivo Glioma-Infiltrating Macrophages

Systemic administration of miR-142-3p resulted in lower macrophageglioma infiltration compared to gliomas treated with scrambled miRNA(FIG. 19A). A negative linear correlation between infiltrating F4/80+macrophages and survival duration in miR-treated animals was observed(R²=0.303, FIG. 19B). More specifically, in a genetically engineeredNtv-a murine model system of high-grade glioma, miR-142-3p treated micealso had extended survival (median survival: 32 days versus 24 days,P=0.03, n=9, FIG. 19A and FIG. 19B). This model system has robustglioma-infiltrating macrophages (21) and treatment with miR-142-3presulted in lower F4/80+ macrophage infiltration compared to the controlgroup (FIG. 19C). A negative linear correlation was established betweenthe infiltrating F4/80+ macrophages and survival in themiR-142-3p-treated mice (R2=0.303, FIG. 19D), indicating that miR-142-3pis exerting a therapeutic effect by modulating glioma-infiltratingmacrophages. miR-142-3p suppressed CD11b+ macrophages elaborating IL-6consistent with M2 skewed cells by 28% (P=0.01) and enhanced CD11b+macrophages elaborating IFN-γ consistent with M1 skewed cells by 83%(P=0.03) within the spleen. In contrast, miR142-3p did not affect thefunctional M1 and M2 composition in blood and lymph nodes (FIG. 24A).However, there was no direct effect of miR142-3p on dendritic cellexpansion, CD8+ effector responses as reflected by the production ofeffector cytokines such as IFN-γ, TNF-α and IL-2, or T-cell mediatedtumor cytotoxicity (FIGS. 24B, 24C and 24D).

To the knowledge of the inventors, this is the first study describing asubtractive approach to identify the preferential expression profile ofmiRNAs within a specific immune population—the tumor-associated, immunesuppressive, alternatively activated macrophage (M2) relative to itsmonocyte precursor. This subtractive screening strategy was intended toclassify those miRNAs that are down regulated by the tumormicroenvironment that should have biological roles in either themonocyte to macrophage differentiation state, skewing to or maintenanceof the M2 phenotype, or play roles in M2-mediated immune suppression. Asa result of the screening process, miR-142-3p emerged as apreferentially down-regulated leading candidate in glioma-associatedmacrophages. Although miR-142-3p has been shown to have a role inregulating immune suppression in the T cell compartment, its biologicalrole in the tumor-associated M2 macrophage has not been previouslydescribed. Without wishing to be bound by any theory, these resultssupport the idea that over expression of miR-142-3p induces selectiveapoptosis in the M2 macrophage population due to the inhibition ofautocrine dependent TGFBR1. The specificity of miR-142-3p for TGFBR1 waspredicted by multiple binding algorithms and confirmed by luciferasereporting assays, and mutational analyses.

It has been previously demonstrated that glioma cancer stem cells caninduce M2 macrophages3 and that this population is a negativeprognosticator in genetically engineered murine model systems ofhigh-grade gliomas (Kong et al., 2010). The specific targeting of the M2immune population in vivo for therapeutic intent is widely recognized asbeing desirable. Prior indirect targeting strategies have includedinhibiting macrophage differentiation and cytokine production (Alavenaet al., 2005), macrophage secretion of MMP-9 (Giraudo et al., 2004), ormacrophage trafficking to the tumor microenvironment (Robinson et al.,2003). On the basis of multiple predictive binding algorithms,luciferase reporting assays, and mutational analyses, it has been showthat miR-142-3p targets the TGFβR1 pathway. This finding was furthersupported by the results of in vitro studies that demonstratedpreferential TGFβR1 pathway inhibition by miR-142-3p in M2 cells anddiminished macrophage infiltration within the glioma microenvironment.It has been found that miR-142-3p induces selective apoptosis in the M2macrophage population due to the inhibition of TGFβR1. This induction ofselective M2 apoptosis by inhibiting the TGFβR1 pathway was furthervalidated with small molecule SB431542 and LY-364947, which areinhibitors of TGFβR1 and by siRNA of TGFβR1. Although LY-364947 also hasinhibitory effects on VEGF (Vogt et al., 2011), which can inducemacrophage to M2 skewing under selective conditions (Linde et al.,2012), preponderant evidence relying on the more specific TGFβR1blockade data (i.e. SB431542 and siRNA), indicates that the induced M2macrophage apoptosis is secondary to inhibition of its dependent TGFβR1pathway. Although autocrine growth dependence on TGF-β has been shown incancer cells (Muraoka et al., 2002), these observations can now beextended to the immune suppressive M2 macrophage. Thus, the demonstratedmechanism of direct targeting of the M2 macrophage by disruptingautocrine TGFβR1 stimulation is also novel and not previously described.

In vivo targeting of TGFβR1, especially in the circulating monocytes asthey become differentiated to the M2 macrophages in glioma-bearing mice,exerted a therapeutic effect against malignant gliomas in a variety ofmodel systems. In vivo therapeutic efficacy has not been demonstratedwith other TGFβR1-targeted therapeutics including the small molecules(Bouquet et al., 2011; Halder et al., 2005; Hjelmeland et al., 2004).The therapeutic effect of miR-142-3p in vivo directly correlated with adecreased glioma macrophage infiltration—specifically the M2 population.The exploitation of the immune system to mediate the therapeutic effectsof miRNA, such as miR-142-3p, can circumvent previous limitations ofmiRNA delivery, including getting past the blood-brain-barrier.Furthermore, circulating immune cells are the first point of contact toadministrated miRNAs—affording an opportunity to directly modulate theirfunctional activity. Despite a therapeutic effect of miR-142-3p againstestablished intracerebral gliomas was evident, therapeutic “cures” werenot frequently observed, especially in the heterogeneous, geneticallyengineered murine models. The heterogeneity of immune suppressivemechanisms and pathways exploited by malignant gliomas is widelyacknowledged and documented. Thus, to target TGFβR1 alone would not beanticipated to result in a cure in clinic. Patients that haveglioblastomas with an enrichment of M2 in the local tumormicroenvironment may be particularly response to treatment withmiR-142-3p.

Treatment with miR142-3p was well tolerated and there was no evidence ofCNS toxicity or induced autoimmunity during administration of miR-142-3pto mice was observed, including demyelination and macrophage/lymphocyteinfiltration in the normal, non-tumor-bearing brain. AlthoughmiR-142-induces preferential apoptosis in the M2 population, theperipheral monocyte counts of treated mice was not affected bymiR-142-3p systemic administration, probably secondary to thepre-existing expression of miR-142-3p in these cells and their lack ofdependency on the TGFβR1 pathway. Moreover, an oncogenic effect ofmiR-142-3p in human T-cell acute lymphoblastic leukemia has beenreported (Lv et al., 2012); however, any significant differences inlymphocyte counts between the miR-142-3p-treated and control mice in theperipheral blood were observed. Reconciliation of these results suggeststhat miR-142-3p plays differential roles (oncopromoter versusoncosuppressor) in different malignancies.

In this study, the following approach to identify miRNA immunetherapeutics using a two-step process was used: 1) screen miRNAexpression from tumor-associated immune cells relative to normal immunecell, and 2) select and prioritize potential candidates on the basis ofbinding to immunosuppressive pathways or mechanisms. Several of thealternative candidates identified in the human miRNA microarrayexpression library may have roles in modulating Treg induction pathwaysand expression of TGF-β, IL-10, CTLA-4, and PD-1. Other candidate miRNAsin this study are being evaluated for their potential as therapeuticagents and could be used in a complementary or alternative fashion withmiR-142-3p. Additionally, miRNAs would be easier and cheaper to producethan antibodies and are not confined to targets on the cell surfacemembrane. As has already been shown the proof of principal that miRNAscan reverse tumor-mediated immune suppression; reciprocally, it isobvious, that miRNAs could be identified by screening the immune cellsfrom patients with autoimmune diseases relative to normal subjects thenselecting the down regulated miRNAs that block pro-inflammatoryresponses. This miRNA identification strategy can be used to identifycancer therapeutics; alternately, this approach may be used to identifynovel therapeutics for autoimmune disorders.

Example 3 miR-138 Exerts Anti-Glioma Efficacy by Targeting ImmuneCheckpoints

A. Materials and Methods

1. Human Glioblastoma Cancer Stem Cells and Glioma Cell Lines

The gCSCs were derived as previously described (Bao et al., 2006) andwere characterization based on the criterion of in vivo tumorigenicpotential, pluripotent potential, limiting dilution assays andcytogenetic characterization which is consistent with our previouslypublished reports (Wei et al., 2010; Wu et al., 2010). The gCSCs werecultured in vitro with neurosphere medium consisting of Dulbecco'smodified Eagle's medium/F-12 medium containing 50 ng/mL of bothepidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2).The murine glioma GL261 cell line was obtained from the National CancerInstitute-Frederick Cancer Research Tumor Repository and maintained inDulbecco's modified Eagle medium (Life Technologies; Grand Island,N.Y.), supplemented with 10% fetal bovine serum (Atlanta Biologicals,Norcross, Ga.), 1% penicillin/streptomycin (Life Technologies), and 1%L-glutamine (Life Technologies). The cells were split every 3 days toensure logarithmic growth. The cells were harvested, washed and thenstained for 30 minutes at 4° C. as described above for the ex vivoglioblastoma tumors.

2. Isolating Total RNA from Glioblastoma

Tumors were pathologically confirmed as glioblastoma (World HealthOrganization grade IV) by a board-certified neuropathologist. Tumorswere washed in RPMI medium and dissected to remove blood products andsurrounding non-tumor brain tissue. The total tissue was broken downinto smaller pieces and digested for 2 hours using a cancer cellisolation kit (Panomics). The cells were suspended in RNAlater solution(Ambion, Austin, Tex.) in RNase Free tubes and stored at 4° C.overnight; after 24 hours, they were transferred to −20° C. until neededfor total RNA extraction. Extraction was performed using the mirVana kit(Ambion). Once extracted, RNA levels were analyzed for concentrationsand purity using UV/Vis spectroscopy at 230, 260, and 280 nm.

3. miR Comparison in Glioblastoma and Normal Brain Tissue

Total RNA extracted from patients was sent to Phalanx Biotech Group(Belmont, Calif.) for microRNA and mRNA-gene expression analyses. TotalRNA of normal brain tissues was obtained from Biochain (Hayward,Calif.). The results of the glioblastoma miR analysis were used todetermine which miRs had significant differences in expression comparedwith normal donor miRs. Fold differences were calculated with MicrosoftExcel. MiRs with the most significant differences in expression levelswere chosen for the miR target analysis using TargetScan (Release 5.1)and RNA22 (FIGS. 25A-D). miRs of interest were selected on the basis ofputative targets and the degree of deviation from normal monocytes.

4. Glioma Tissue Microarrays

This analysis was conducted according to the MD Anderson approvedLAB09-0463, which includes one set of tumor microarrays (TMA) ofpatients with different glioma grades and pathologies and is designated“glioma” TMA and includes WHO grade IV glioblastomas (n=53), WHO gradeIII anaplastic astrocytomas (n=17), WHO grade II low-grade astrocytomas(n=3), WHO grade II oligodendrogliomas (n=16), WHO grade III anaplasticoligodendrogliomas (n=15), WHO grade II mixed oligoastrocytomas (n=6),WHO grade III anaplastic mixed oligoastrocytomas (n=12), and WHO gradeIV gliosarcomas (n=7) and has been previously described (Barnett et al.,2007). A second glioblastoma-specific TMA was constructed underPA12-0136 and contains 99 glioblastomas. For TMA construction, two 1-mmcores were obtained per tumor sample. The rationale for using a TMA wasto facilitate an analysis of the largest number of tumor samplespossible. The study neuropathologist (G.N.F.) gathered the tissuesections from the archived paraffin blocks and confirmed the tumorpathologic type. The time from resection to fixation was less than 20minutes in all cases, in accordance with the Clinical LaboratoryImprovement Amendments standard.

In situ hybridization was performed using the protocol developed byNuovo et al (Nuovo et al., 2009), with some minor adjustments as we havepreviously described (Wei et al., 2013). Digoxigenin-labeled, lockednucleic acid-modified probes for miR-138 (hsa-miR-138) and the positivecontrol (U6, hsa/mmu/rno) were purchased from Exiqon (Vedbek, Denmark)and were used for detecting miR-138 expression on the TMA. Thecolorimetric reaction was monitored visually and stopped by placing theslides in water when background coloring started to appear. The TMA wasanalyzed by the study neuropathologist (G.N.F.). In the assessment ofmiR-138 expression in gliomas, intervening neurons in the infiltratingcomponent were not considered positive.

5. miR Relative Expression Survival Analysis

Complete data for 383 glioblastoma patients that included expression ofmiR-138 and PD-L1, together with survival data, was downloaded from TheCancer Genome Atlas (TGCA). The Kaplan-Meier survival curve data wasplotted for the top 50% versus bottom 50% miR-138 expression and top 20%versus lowest 20% expression using GraphPad Prism5 (GraphPad Software,Inc., La Jolla, Calif.).

6. PD-L1 Expression in Glioblastomas

The glioblastoma TMA was de-paraffinized in xylene and rehydrated inethanol. We used a commercial Abcam (Cambridge, Mass., USA) polyclonalanti-PD-L1 antibody. The slides were de-paraffinized in xylene andrehydrated in ethanol. Antigen retrieval was carried out for 30 minutesin citric acid buffer (pH 6.0). Endogenous peroxidase activity wasblocked with immersion in 0.3% hydrogen peroxide in methanol for 30minutes. Slides were blocked with 1:100 normal goat serum for 20 minutesat room temperature and then they were incubated with the primaryantibody at a 1:200 dilution at 4 degrees overnight. The slides wereincubated with a goat anti-rabbit secondary antibody at 1:200 for onehour, and then with the avidin/biotin complex (Vectastain ABC kit,Vector Laboratories, Burlingame, Calif., USA) at 1:100 for one hour.Visualization was performed with the chromagen DAB (Sigma-Aldrich Corp.,St Louis, Mo., USA), slides were counterstained with hematoxylin,dehydrated and mounted. The TMA was inspected under 200× magnificationand the positive stained cells were quantified under the directsupervision of the study neuropathologist.

To secondarily validate these findings, glioblastoma surgery specimenswere processed within 4 h after resection. To detect PD-L1 expression onex vivo glioblastoma cells by flow cytometry analysis, the 10F.9G2 clonewas used (Biolegend, San Diego, Calif.). Briefly, the tumor wasmechanically dissociated, enzymatically digested for one hour inLiberase™, passed sequentially through 100 and then 70 micron filters,incubated in red cell lysis buffer for 15 minutes, then spun, blockedand then stained with the PD-L1 antibody or isotype control for 20minutes at 4° C. Data acquisition was performed using the BeckmanCoulter Gallios flow cytometer (Beckman Coulter, Brea, Calif.). Analysiswas performed using FlowJo software.

7. Luciferase Assay

To determine whether miR-138 can bind to CTLA-4 3′-UTR and or PD-13′UTR, H9 (ATCC, Manassas, Va.) cells were co-transfected with thedesignated luciferase reporter plasmid (pMirTarget, Origene, Rockville,Md.) and miR-138 expression plasmid or scramble control plasmid(GeneCopoeia) with Lipofectamine 2000 transfection reagent (Invitrogen).A control set of H9 cells were transfected with the luciferase reporterplasmid without the addition of a 3′ UTR to evaluate for off-targeteffects of miRNA-138 on the reporter plasmid itself. Renilla luciferasereporter plasmid was included as an internal control for transfectionefficiency. The interaction between miR-138 and its target were measuredby comparing the results of the co-transfection of the CTLA-4 3′ UTR, orPD-1 3′-UTR-luciferase reporter and miR-138 plasmids with those of the3′ UTR-luciferase reporter plasmid and the scramble control plasmid. Wealso transfected HeLa reporter cells and performed the same analysiswith mutant CTLA-4 and PD-1 3′ UTR luciferase reporter plasmids. Foreach mutant, we changed 5 base-pairs; one mutant each was generated forthe miR-138 binding site of PD-1, one mutant was created for each of themiR-138 binding sites of CTLA-4, and one mutant was created with allthree binding sites mutated. The luciferase assay was performed usingthe Dual-Luciferase® reporter assay system (Promega, E1910). Fireflyluciferase activity was normalized by renilla luciferase activity.

8. In Vivo Experiments

The miR-138 duplex that mimics pre-miR-138 (sense:5′-AGCUGGUGUUGUGAAUCAGGCCGU-3′ (SEQ ID NO:20), antisense:5′-GGCCUGAUUCACAACACCAGCUGC-3′(SEQ ID NO:21)) and the scramble controlmiRNA duplex (sense: 5′-AGUACUGCUUACGAUACGGTT-3′ (SEQ ID NO:22),antisense: 5′-CCGUAUCGUAAGCAGUACUTT-3′ (SEQ ID NO:23)) were synthesized(SynGen, San Carlos, Calif.). The sequence of murine miR-138 isidentical to human miR-138 on the basis of NCBI blast data. Thetreatment cohorts consisted of 2 μL miR-138 or scramble control (10μg/+48 μL of phosphate-buffered saline (PBS) mixed with the vehicle (40μL PBS+10 lipofectamine 2000) or the vehicle control (90 μL PBS+10 μLlipofectamine 2000). Mice were maintained in the MD Anderson IsolationFacility in accordance with Laboratory Animal Resources Commissionstandards and conducted according to the approved protocol 08-06-11831.

9. Syngeneic Subcutaneous Model

The B16 cells were kindly provided by Dr. Willem Overwijk (theUniversity of Texas M.D. Anderson Cancer Center, Houston, Tex.) and weremaintained in RPMI 1640 medium supplemented with 10% FBS at 370 C in ahumidified atmosphere of 5% CO2 and 95% air. The murine glioma GL261cell line was obtained from the National Cancer Institute-FrederickCancer Research Tumor Repository and maintained in Dulbecco's modifiedEagle medium (Life Technologies; Grand Island, N.Y.), supplemented with10% fetal bovine serum (Atlanta Biologicals, Norcross, Ga.), 1%penicillin/streptomycin (Life Technologies), and 1% L-glutamine (LifeTechnologies). The cells were split every 3 days to ensure logarithmicgrowth.

To induce subcutaneous tumors, logarithmically growing GL261 cells wereinjected into the right hind flanks of 6-week-old C57BL/6J female miceor nude mice at a dose of 1×106 cells suspended in 100 μl of PBS dilutedmatrigel basement membrane matrix (BD Biosciences) (PBS:Matrigel=2:1).When approximately 0.5 cm palpable tumors formed, the mice (n=5/group)were treated by local tumor or intravenous injection. Tumors weremeasured twice a week. Mice that showed signs of morbidity, high tumorburden, or skin necrosis were immediately euthanized according to MDAnderson guidelines. Tumor volume was calculated with slide calipersusing the following formula: V=(L×W×H)/2, where V is volume (mm³), L isthe long diameter, W is the short diameter, and H is the height.

10. Syngeneic Intracranial Glioma Model

To induce intracerebral tumors in C57BL/6J mice and athymic nude mice,GL261 cells or B16 cells were injected into the cerebrum. These cellswere collected in logarithmic growth phase, washed twice with PBS, mixedwith an equal volume of 10% methyl cellulose in Improved modifiedEagle's medium Zinc Option medium, and loaded into a 250-μ1 syringe(Hamilton, Reno, Nev.) with an attached 25-gauge needle. The needle waspositioned 2 mm to the right of bregma and 4 mm below the surface of theskull at the coronal suture using a stereotactic frame (KopfInstruments, Tujunga, Calif.), as we previously described (Heimberger etal., 2003). The intracerebral tumorigenic dose for GL261 cells was 5×104and for B16 was 5×102 in a total volume of 5 μl. Mice were then randomlyassigned to control and treatment groups (n=10/group for GL261,n=6-7/group for B16). Animals were observed three times per week, andwhen they showed signs of neurological deficit (lethargy, failure toambulate, lack of feeding, or loss of >20% body weight), they werecompassionately euthanized. These symptoms typically occurred within 48hours of death. The brains were removed and placed in 4%paraformaldehyde and embedded in paraffin.

11. Genetically Engineered Murine Model of Glioma

We have previously described the use of immune competent Ntv-A mice withRCAS-PDGFB+RCAS-Bcl-2 induced high-grade gliomas for testing immunetherapeutics (Kong et al., 2010). Briefly, the transgenic Ntv-a mice aremixtures of different strains, including C57BL/6, BALB/c, FVB/N, andCD1. PDGFB and Bcl-2 are transferred into the cerebral hemispheres ofmice by injection of RCAS vectors containing these genes at the coronalsuture on postnatal days 1 or 2. The mice are randomized to treatmentstarting on day 21 with anti-CTLA-4 antibody (clone 9H10), isotypecontrol, miR-138, or scramble control. The miR-138 treatment wasinjected on Monday, Wednesday and Friday for three weeks, while theanti-CTLA-4 was delivered by intraperitoneal injections on day 21, 24and 27 as previously described (Fecci et al., 2007). The mice weremonitored for morbidity from tumor burden, and were euthanized ifpresent. If not, all mice were euthanized at 90 days, the brains wereremoved, and analyzed for tumor formation and grading by the studyneuropathologist.

12. Immunohistochemistry of Treated Gliomas

Formalin-fixed, paraffin-embedded sections of the treated and untreatedGL261-implanted brains and the Ntv-A mice harboring gliomas weredeparaffinized in xylene and rehydrated in ethanol. Antigen retrievalwas performed by immersing the sections in a citrate-buffered solution(pH 6.0), heating them in the microwave for 2 minutes, then maintainingheat by placement in a steamer for 30 minutes. The samples were thencooled to room temperature. Endogenous peroxidase was blocked with 0.3%hydrogen peroxide/methanol for 30 min at room temperature. Afterblocking with a protein block serum-free solution (DAKO, Carpinteria,Calif.), diluted antibody anti-FoxP3 antibody (eBioscience) was applied.The samples were then washed and incubated with the appropriatesecondary antibody for one hour. They were then washed and incubatedwith streptavidin for one hour, and then each sample was developed usingthe SigmaFAST DAB kit (Sigma-Aldrich, St Louis, Mo.). Color developmentwas stopped by gently dipping the slides in distilled water. The nucleiwere then counterstained with hematoxylin.

13. Isolation of Human CD4+ T Cells

Human PBMCs were prepared from blood donated by healthy volunteers andglioblastoma patients undergoing resection at The University of Texas MDAnderson Cancer Center (Houston, Tex.). The PBMCs were isolated bycentrifugation on a Ficoll-Hypaque density gradient (Sigma-Aldrich, St.Louis, Mo.) and the CD4+ T cells were purified by negative selectionmicrobead magnetic cell sorting according to the manufacturer'sinstructions (BD Biosciences, San Jose, Calif.).

14. miR-138 Transfection in T Cells

CD4+ T cells purified from healthy donor PBMCs at 0.5 million/ml wereplated on anti-CD3/anti-CD28 antibody (BD Biosciences, San Jose, Calif.)pre-bound 24-well plates for 48 hours for TCR activation and cellproliferation either with or without the addition of 5 ng/ml TGF-β. CD4+cells were then harvested, washed and transfected with miR-138 orscramble RNA expressing vectors (GeneCopoeia, Rockville, Md.) via theNucleofector human T cells transfection kit (Lonza, Allendale, N.J.;program: T-023).

15. Functional Analysis of T Cells

CD4+ cells transfected with miR-138 or scramble RNA-expressing vectors,with or without activation by TGF-β, as above, were plated into 96 wellplates in 100 ul samples. Cells were surface stained with antibodies toPD-1 (conjugated to PercP, from eBioscience) and CTLA-4 (conjugated toPE, from BD) and permeabilized for intracellular staining of FoxP3(conjugated to APC, from eBioscience). Samples were then evaluated byflow cytometry. The samples were gated for live cells, and theexpression of PD-1, CTLA-4 and FoxP3 were evaluated. For ICOSdeterminations, CD4+ cell enrichment, transfection, activation andplating were as above. Cells were then permeabilized for ICOS staining(conjugated to APC, eBioscience 17-9948-42) and evaluated by flowcytometry.

16. Real-Time PCR to Confirm Relative miR-138 Expression Levels

Total RNA extracted from T cells, including those transfected withmiR-138, was used as the template for reverse transcription using theTaqMan reverse transcription kit (Applied Biosystems, Carlsbad, Calif.)in a thermocycler per the manufacturer's instructions. Primers forreverse transcription were purchased for human miR-138 and U18 (AppliedBiosystems). U18 was used as an endogenous control. cDNA was used as thetemplate for real-time PCR. U18 and miR-138 amplifications were run induplicate using the TaqMan real-time PCR kit (Applied Biosystems) in the7500 real-time PCR system (Applied Biosystems). Further reactions,substituting water for the cDNA template, were used as additionalcontrols. Excel was used to calculate the mean levels of each miR andthe U18 internal control. The relative expression levels of miR-138 werecompared with those of the internal controls.

17. Statistics

Survival differences on Kaplan-Meier curves were calculated in theGraphPad Prism5 software using the Log-rank (Mantel-Cox) test.

B. Results

1. miR-138 Expression in Glioblastoma

To determine the pattern of miR expression in glioblastoma relative tonormal brain tissue, we used the Human miRNA OneArray Microarray v2 (Weiet al., 2013). The identified down-regulated miRs were then screened forpotential targeting to the immune checkpoints CTLA-4 and PD-1 usingRNA22. miR-138 emerged as a leading candidate with a mean 13.4-folddecrease in expression from normal brain tissue and with three bindingsites in the 3′ UTR of CTLA-4 and one within in PD-1. A subsequentanalysis using reverse transcription-polymerase chain reaction (RT-PCR)confirmed that miR-138 was absent or minimally expressed in glioblastomaspecimens (mean=0.20; range: 0.003 to 0.65; n=3) and glioma cell lines(mean=0.28; range: 0.10 to 0.45; n=4) relative to normal brain tissues(mean=3.9; range: 1.7 to 7.0; n=3). Using a glioma tumor microarray andin situ hybridization, heterogeneous expression of miR-138 was foundamongst all glioma grades and pathological subtypes (Table 7). Nodifference in survival time among glioblastoma patients was found on thebasis of the relative expression of miR-138 in The Cancer Genome Atlasdata set (FIGS. 29A-B) consistent with data from a previously publishedreport (Chan et al., 2012).

TABLE 7 miR-138 Expression in Gliomas WHO n grade − + ++ +++ Mixedoligoastrocytoma 6 II 0 50 50 0 Oligodendroglioma 25 II 0 48 48 4Low-grade astrocytoma 3 II 0 100 0 0 Anaplastic oligodendroglioma 16 III5 57 25 13 Anaplastic mixed oligodendroglioma 12 III 0 25 58 17Anaplastic astrocytoma 40 III 12 58 28 2 Gliosarcoma 11 IV 0 46 54 0Glioblastoma 87 IV 8 53 31 8

2. miR-138 Inhibits Tregs by Targeting Immune Checkpoints

To validate the targets prove that miR-138 binds to the immunecheckpoint molecules, luciferase expression assays were conductedincluding mutating the predicted CTLA-4 and PD-1 3′-UTR binding sites ofmiR-138 (FIG. 25A-B). PD-1 luciferase activity was significantlyinhibited by 18±5.0% by miR-138; whereas mutational alteration of themiR-138 PD-1 3′-UTR binding site abolished the inhibitory effect of themiR-138 on PD-1 expression (FIG. 25C).

To determine the effects of miR-138 on the de novo synthesis of Tregs,miR-138 was transfected into human T cells during in vitro Treginduction with TGF-β1. miR-138 transfected T cells significantlydown-regulated CTLA-4, PD-1 and FoxP3 expression compared to thescramble control (FIG. 26A-C). Since the mechanism of activity ofipilimumab has been shown to entail the activation of the ICOS/ICOSLpathway (an inducible co-stimulator) (Fu et al., 2011; Carthon et al.,2010; Chen et al., 2009) and the ICOS ligand is expressed by gliomacells (Schreiner et al., 2003), we evaluated if miR-138 would alter ICOSexpression in human T cells but found that there was no difference aftermiR-138 transfection versus scramble control stimulated andun-stimulated CD4+ cells.

3. miR-138 Inhibits In Vivo Glioma Growth

Given the role of miR-138 in modulating the CTLA-4 and PD-1 pathways, wenext determined whether miR-138 exerted a therapeutic effect in vivo. Toassess the in vivo anti-tumor efficacy of miR-138, GL261 murine gliomacells were implanted into immune competent C57BL/6 mice and were treatedwith miR-138 or scramble control (n=10 per group). After thesubcutaneous GL261 tumors had grown to a palpable size, miR-138 duplexor scramble control was administered. Subcutaneous tumor growthprogressed in all the C57BL/6J mice treated with the scramble control.In contrast, in the miR-138-treated group, the tumor volume was markedlysuppressed (P<0.05) (FIG. 27A). Gliomas started to shrink as soon asmiR-138 was administered; moreover, the tumors continued to regress evenafter miR-138 treatment was discontinued. In contrast, tumors keptgrowing aggressively in scramble microRNA-treated and untreatedtumor-bearing mice groups.

To ascertain if systemic administration of miR-138 had a therapeuticeffect against established intracerebral gliomas, C57/BL6J mice withestablished GL261 tumors were treated with i.v. administered miR-138. Inmice treated with miR-138 median survival was 33.5 days which comparedfavorably to mice treated with scramble control with a median survivalof 23.5 days (P=0.011) (FIG. 27B). In the aggressive B16 murine model ofintracranial melanoma, treatment with miR-138 increased median survivalby 23%.

4. The Therapeutic Effect of miR-138 is Immune Mediated

To determine if the therapeutic effect of miR-138 is immunologicallymediated, nude mice were implanted with GL261 and subsequently treatedwith miR-138. In the immune-incompetent animal background, miR-138failed to exert any therapeutic effect, indicating that miR-138 mediatesin vivo activity via the immune system (FIG. 28A). Ex vivo analysis ofthe miR-138-treated GL261 gliomas from the immune competent micedemonstrated a marked reduction of FoxP3+ T cells by 51% relative to thescramble control (P=0.03) (FIG. 28B).

5. The PD-1 Ligand, PD-L1, is Expressed in Glioblastomas

To determine if the overexpression of PD-L1 impacts the survival ofglioblastoma patients, we analyzed expression relative to outcome usingthe TCGA database. The relative expression levels of PD-L1 werecategorized by the upper and lower percentiles of expression, and therewas a statistically significant difference between survival, with thepatients with PD-L1 expression in the top 20^(th) percentile survivinglonger than those in the bottom 20% of relative expression (FIG. 30B).To validate this data, the frequency of PD-L1 expression in gliomas andits prognostic impact were determined by immunohistochemical stainingfor PD-L1 on the glioblastoma TMA (FIG. 30A). We found that universallythat almost all glioblastomas express PD-L1 expression. Furthermore, bydirect ex vivo staining of glioblastomas by flow cytometry, mostdemonstrated positive PD-L1 expression. Since have previouslydemonstrated the glioblastoma cancer stem cells (gCSCs) are profoundlyimmune suppressive and since these cells can be infrequent within theoverall tumor mass, we analyzed the expression of PD-L1 expression ongCSCs and found only low frequency of expression on rare gCSCs. However,PD-L1 is expressed on commonly used laboratory glioma cell lines such asGL261 and U87 (FIG. 30C).

Unlike many cancers such as melanoma, where immune therapy has madegreat strides over the years, immunotherapy for glioma has not yetmatured into therapeutics capable of successful widespread translationto the clinic (Heimberger et al., 2011). Many reasons exist for this,including difficulties in scaling up production for many difficult andexpensive processes as is the case of dendritic cell vaccines,challenges in clinical trial design, lack of interest in drugdevelopment for glioblastoma by pharmaceutical companies secondary tothe rarity of the disease and barriers in drug delivery. Use of themicroRNAs described herein may present an opportunity to sidestep manyof these concerns. Their easy reproduction lowers costs, as above, andtheir biocompatibility may lower toxicity concerns. As shown in theabove examples, two other miRNAs (i.e., miR-124 and miR-142-3p) downregulated in glioblastoma may be used therapeutically, e.g., byovercoming immune suppression by different mechanisms. Given thevariable expression of immunosuppressive cells throughout the differentsubtypes of glioblastoma (Doucette et al., 2013, these miRNAs may becombined (e.g., miR-124, miR-142-3p, and/or miR-138) for furthertherapeutic synergy and comprehensive targeting of tumor-mediated immunesuppression.

Example 4 Combination Therapies Involving miR-138 miR-142-3p, and/ormiR-138

To assess whether these immune modulatory miRNAs would have an additiveor synergistic therapeutic effect, we assessed all possible combinationsand compared to monotherapy. C57BL/6J mice with establishedintracerebral GL261 gliomas were treated intravenously for 3 weeks onMonday, Wednesday, and Friday with 20 μg of each miRNA (scramblecontrol, miR-124, miR-138, miR-142-3p, miR124+miR142-3p, miR124+miR138,miR142-3p+miR138, miR124+miR138+miR142-3p) in 48 μL ofphosphate-buffered saline (PBS) mixed with the vehicle (40 μL PBScontaining 10 μL LIPOFECTAMINE₂₀₀₀₎.

Results are shown in FIG. 31. The study endpoint is median survivaltime. miR-124 was identified as the lead monotherapy andmiR-138+miR142-3p the optimal combinatorial approach. More specifically,it appears that the CTLA-4 and PD-1 inhibition provided by miR-138 issynergistic with the TGFBR1 inhibition in immunosuppressive M2macrophages provided by miR-142-3p. Complementary mechanistic studiesare underway that will evaluate both the systemic and intratumoralimmune responses that are modulated with this combinatorial approach.

Example 5 Formulations of Immune Modulatory miRNAs for Use in CancerPatients

We have conducted a number of formulation equivalency studies withmiRNAs and found that co-administration with DOTAP was not satisfactoryin comparison to LIPOFECTAMINE. MiR-124 chitosan nanoparticles wereprepared on an ionic gelation of anionic tripolyphosphate. Furthermore,miR-124 DOTAP:cholesterol nanoparticles were created. Based on efficacyagainst intracranial GL261 glioma tumors, these constructs were notdeemed to be sufficiently satisfactory for further translationaldevelopment (FIG. 32). Further modifications were made to enhance thetherapeutic delivery of miRNAs to the immune cell population.

Specific formulation modifications were made in the lipid nanoparticlesto potentially drive entry into the circulating immune cell populationand to accommodate the miRNAs. To ascertain the pharmacokinetics andhalf-life of liposomes comprising miR-124 in vivo, non-tumor bearingC57BL/6J mice may be dosed at 1 mg/kg of the test article and the PBMC,liver and serum will be fractionated at various time points. Threeanimals will be used per each experimental time point of 0, 15 minutes,1, 4, 8 and 24 hours. The comparator (positive control) will be miR-124administered with LIPOFECTAMINE for which we have previously conductedthis type of analysis (FIG. 32B). The miR-124 duplex that mimicspre-miR-124a (sense: 5′-UAAGGCACGCGGUGAAUGCCA-3′ (SEQ ID NO:4),antisense: 3′-UAAUUCCGUGCGCCACUUACG-5′ (SEQ ID NO:5)) is synthesized bySynGen, San Carlos, Calif. The treatment cohorts consisted of 20 μg ofthe miR-124 duplex in 10 μL of PBS mixed with the vehicle (80 μL PBScontaining 10 μL LIPOFECTAMINE 2000; Invitrogen). Quantitative PCR maybe used to assess the miRNA level in each compartment and it's in vivohalf-life, similar to the previous analysis we have conducted formiR-124+LIPOFECTAMINE (FIG. 33).

Experiment 2:

Ascertain if liposomes targeting the STAT3 pathway and has select immunemodulatory properties. The inventors have shown that miR-124 can targetmultiple hubs within the STAT-3 signaling pathway. The levels of bloodand spleen T cells have only modest expression of p-STAT3 even in tumorbearing animals. The highest levels of p-STAT3 were observed within thetumor microenvironment (FIG. 34).

C57BL/6J mice bearing subcutaneous GL261 cells may be treated by eithermiR-124+LIPOFECTAMINE 2000 (n=3 per group) or control liposomes for twoweeks. For glioma-infiltrating T-cell isolation, the gliomas may be cutinto small pieces and digested with Liberase™ for 2 hours at 37° C. tomake a single-cell suspension for CD3+ T-cell selection using the CD3negative selection kit (BD Biosciences). The purity of CD3+ T-cellsis >94%. For intracellular p-STAT3 detection, the CD3+ T cells are firstfixed with 2% paraformaldehyde at room temperature for 10 minutes.Thereafter, the cells were washed and permeabilized with 90% methanol onice for 30 minutes, and then stained with PE-conjugated anti-p-STAT3(Y705) antibody (BD Pharmingen) for 30 minutes at room temperature. Themost robust immune modulatory endpoint was the inhibition of Tregs inthe glioma microenvironment, and p-STAT3 is a transcriptional regulatorof FoxP3. As a secondary endpoint, the T cells may also be stained witha PE-conjugated anti-FoxP3 antibody. Flow cytometry acquisition may beperformed with a FACS CALIBUR (Becton Dickinson, San Diego, Calif.), anddata will be analyzed with FLOWJO software (TreeStar, Ashland, Oreg.).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 5,034,506-   U.S. Pat. No. 5,235,033-   U.S. Pat. No. 5,399,363-   U.S. Pat. No. 5,466,468-   U.S. Pat. No. 5,466,468-   U.S. Pat. No. 5,543,158-   U.S. Pat. No. 5,629,001-   U.S. Pat. No. 5,641,515-   U.S. Pat. No. 5,756,353-   U.S. Pat. No. 5,780,045-   U.S. Pat. No. 5,804,212-   U.S. Pat. No. 6,268,490-   U.S. Pat. No. 6,613,308-   U.S. Pat. No. 6,770,748-   U.S. Patent Publication No. 2002/0115080-   U.S. Patent Publication No. 2005/0107325-   U.S. Patent Publication No. 2005/0182005-   Allavena et al., Cancer Res. 2005; 65:2964-2971.-   Bao et al., Nature. 2006; 444:756-60.-   Barnett et al., Clin Cancer Res. 2007; 13:3559-67.-   Behm-Ansmant et al., Cold Spring Harb. Symp. Quant. Biol.,    71:523-530, 2006.-   Bharali et al. PNAS (2005) 102(32): 11539-11544-   Bjorge et al., PLoS One. 2011; 6:e19309.-   Bouquet et al., Clin Cancer Res. 2011; 17:6754-6765.-   Brennecke et al., Cell, 113(1):25-36, 2003.-   Cai et al., Stem Cells. 2012; 30:1746-55.-   Calin et al., Proc. Natl. Acad. Sci. USA, 105:5166-5171, 2008.-   Carrington and Ambros, Science, 301(5631):336-338, 2003.-   Carthon et al., Clin Cancer Res. 2010; 16:2861-71.-   Chan et al., Cancer Res. 2005; 65:6029-33.-   Chan et al., Cell Cycle. 2011; 10:1845-1852.-   Chan et al., Cell Rep. 2012; 2:591-602.-   Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987.-   Chen et al., J Leukoc Biol. 2008; 83:1165-1173.-   Chen et al., Mol. Microbiol., 53843-856, 2004.-   Chen et al., PLoS ONE, 6, 2011.-   Chen et al., Proc Natl Acad Sci USA. 2009; 106:2729-34.-   Chen, Science, 303:2022-2025, 2004.-   Chihara et al., J Immunol. 2012; 188:3620-3627.-   Chu and Rana, Plos. Biology., 4:e210, 2006.-   Corsten et al., Cancer Res. 2007; 67:8994-9000.-   Dai et al., Cancer Res. 2011; 71:3658-68.-   Dai et al., Genes Dev. 2001; 15:1913-1925.-   Dostie et al., Rna-A Publication of the Rna Society, 9:180-186,    2003.-   Doucette et al., Cancer Immunol Res. 2013; 1:112-22.-   Doucette et al., Neuro Oncol., 2012; 14:1136-45.-   Eulalio et al., Cell, 132:9-14, 2008.-   Fecci et al., Clin Cancer Res. 2007; 13:2158-67.-   Flavell et al., Nat Rev Immunol. 2010; 10:554-567.-   Friedman et al., Genome Res., 19:92-105, 2009.-   Fu et al., Cancer Res. 2011; 71:5445-54.-   Gabriely et al., Mol Cell Biol. 2008; 28:5369-80.-   Gaur et al., Neuro Oncol. 2011; 13:580-90.-   Giraudo et al., J Clin Invest. 2004; 114:623-633.-   Halder et al., Neoplasia. 2005; 7:509-521.-   Hardee et al., Cancer Res. 2012; 72:4119-4129.-   Hashimoto et al., Blood. 1999; 94:837-844.-   Hatziapostolou et al., Cell. 2011; 147:1233-47.-   Heimberger et al., Clin Cancer Res. 2003; 9:4247-4254.-   Heimberger et al., Neuro Oncol. 2011; 13:3-13.-   Heusinkveld et al., J Transl Med. 2011; 9:216.-   Hjelmeland et al., Mol Cancer Ther. 2004; 3:737-745.-   Hui et al., Clin Cancer Res. 2010; 16:1129-1139.-   Huse et al., Brain Pathol. 2009; 19:132-143.-   Hussain et al., Neuro Oncol. 2006; 8:261-279.-   Iliopoulos et al., Mol Cell. 2010; 39:493-506.-   Inman et al., Mot Pharmacol. 2002; 62:65-74.-   Inoue et al., Oncol Rep. 2012; 27:1759-1764.-   Jarkowski et al., J Oncol Pharm Pract. 2013.-   Jensen et al., J Clin Oncol. 2009; 27:3330-3337.-   Kong et al., Clin Cancer Res. 2010; 16:5722-33.-   Kortylewski et al., Nat Biotechnol. 2009; 27:925-32.-   Kortylewski et al., Nat Med. 2005; 11:1314-21.-   Krausgruber et al., Nat Immunol. 2011; 12:231-238.-   Krutzfeldt et al., Nature, 438(7068):685-689, 2005.-   Lim et al., Nature. 2005; 433:769-73.-   Linde et al., J Pathol. 2012; 227:17-28.-   Loffler et al., Blood. 2007; 110:1330-3.-   Lu et al. (Cancer Cell (2010) 18:185-197)-   Lujambio et al., Cancer Res. 2007; 67:1424-9.-   Lv et al., Leukemia. 2012; 26:769-777.-   Magrassi et al., Oncogene. 2005; 24:5198-206.-   Metzler et al., Genes Chromosomes Cancer, 39:167-169, 2004.-   Michael et al., Mol. Cancer Res., 1:882-891, 2003.-   Muraoka et al., J Clin Invest. 2002; 109:1551-1559.-   Murray et al., Biochem Soc Trans. 2006; 34:1028-31.-   Namlos et al., PLoS One. 2012; 7:e48086.-   Nuovo et al., Nat Protoc. 2009; 4:107-15.-   Onco-Path siRNA Library—Overview. 2013; Available from:    avetrabiocom.ipage.com/1701.html-   Pander et al., Clin Cancer Res. 2011; 17:5668-5673.-   Pfeffer et al., Science, 304:734-736, 2004.-   Pinheiro et al., Science, 336(6079):341-344, 2012.-   Ponomarev et al., Nat Med. 2011; 17:64-70.-   Pramanik et al. Mol Cancer Ther (2011) 10:1470-1480-   Puig-Kroger et al., Cancer Res. 2009; 69:9395-9403.-   Reinhart et al., Nature, 403:901-906, 2000.-   Robinson et al., Cancer Res. 2003; 63:8360-8365.-   Schreiner et al., GLIA. 2003; 44:296-301.-   Scuto et al., Cancer Res. 2011; 71:3182-8.-   Seth et al., J Med. Chem., 52(1): 10-13, 2009.-   Seth et al., Nucleic Acids Symp. Ser. (Oxford), 52:553-554, 2008.-   Sierra-Filardi et al., Blood. 2011; 117:5092-5101.-   Soutschek et al., Nature, 432:173-178, 2004.-   Stupp et al., N Engl J Med. 2005; 352:987-996.-   Teplova et al., Nat. Struct. Biol., 6:535-539, 1999.-   Veedu et al., Bioorganic Med. Chem. Lett., 20(22):6565-6568, 2010.-   Vogt et al., Cell Signal. 2011; 23:1831-1842.-   Wang et al., Mot Biol Rep. 2012; 39:2713-2722.-   Wei et al., Cancer Res. 2013; 73:3913-26.-   Wei et al., Clin Cancer Res. 2010; 16:461-73.-   Wei et al., Mol Cancer Ther. 2010; 9:67-78.-   Wu et al., FEBS Lett. 2011; 585:1322-1330.-   Wu et al., Neuro Oncol. 2010; 12:1113-25.-   Xu et al., Curr. Biol., 13(9):790-795, 2003.-   Zhao et al., Cell, 129:303-317, 2007.-   Zhou et al., Lab Invest. 2010; 90:144-55.

1-32. (canceled)
 33. A pharmaceutical preparation comprising miR-124,miR-142, or miR-138.
 34. The pharmaceutical preparation of claim 33,wherein the miRNA comprises a phosphoramidate linkage, aphosphorothioate linkage, a phosphorodithioate linkage, or anO-methylphosphoroamidite linkage.
 35. The pharmaceutical preparation ofclaim 33, wherein the miRNA is a LNA.
 36. The pharmaceutical preparationof claim 33, wherein the pharmaceutical preparation is formulated forintravenous, intraperitoneal, intratumoral, intrathecal, intranasal,intralymphatic, or oral administration.
 37. The pharmaceuticalpreparation of claim 33, wherein the miRNA is comprised in a liposome,nanoparticle, or exosome.
 38. The pharmaceutical preparation of claim33, wherein the pharmaceutical preparation comprisesN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate(DOTAP) or Lipofectamine™. 39-45. (canceled)
 46. The pharmaceuticalpreparation of claim 37, wherein the miRNA is comprised in ananoparticle.
 47. The pharmaceutical composition of claim 38, whereinthe pharmaceutical preparation comprises Lipofectamine™.
 48. Thepharmaceutical composition of claim 36, wherein the pharmaceuticalpreparation is formulated for intravenous administration.
 49. Thecomposition of claim 37, wherein the miRNA is comprised in a liposome.50. The composition of claim 49, wherein the liposome comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),dimyristoylphosphatidylglycerol (DMPG), polyethylene glycol (PEG), orcholesterol.